Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR DETERMINING TRANSCRIPTION FACTOR ACTIVITY
AND ITS TECHNICAL USES
FIELD OF THE INVENTION
This invention relates to the field of molecular biology. In
particular, this invention relates to transcription factors, methods of
analyzing transcription factor activity, and to various technologies to
which methods of analyzing transcription factor activity are related.
BACKGROUND OF THE INVENTION
Rapid advances in DNA technology have created a swell of new
products and therapies through biotechnology, but the greatest gap in
our ability to manipulate biological systems has been in the area of
controlling gene expression. Specifically, despite our understanding and
mapping of nearly all gene coding regions in human and several other
species, we cannot 'decode' the DNA sequence that lies upstream of a
gene, called the promoter. Enhancer binding proteins (subsequently
referred to as transcription factors) bind to these regions, typically more
than a dozen different ones bind to each and every gene promoter.
How these factors work together to so precisely control the expression
of genes has remained unsolved.
At stake is having predictive power of when a gene will be turned
on or off based on assessment of the gene specific transcription factors
present in a living cell at any given time, and in response to any
treatments, including drugs. Also, the ability to alter gene expression or
create promoters starting with individual transcription factor binding sites
has been greatly impaired because one cannot 'read' what these factors
are doing.
Currently, despite identifying many individual transcription factors,
and in some cases having cloned them to know their exact amino acid
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sequences, how these factors help to catalyze transcription is not
known. The current l:hinking is turning towards alterations in chromatin
structure mediated by transcription factors possessing histone acetylase
or deacetylase activity, yet most of the transcription factors positioned
upstream of an eukaryotic gene do not have these activities.
Much work in t:he past in the transcription field has been focused
on the role of so called 'general transcription factors' (GTFs), i.e., those
factors required by all promoters to achieve gene transcription (see, for
example, the work of Roeder, Sharp, Tjian, and Reinberg). The
upstream binding transcription factors that control transcription rates
have been presumed to interact with the components of the general
transcription complex. In the dominant paradigm, a direct interaction
befinreen these upstream transcription factors and the GTFs presumably
occurs by looping out of the intervening DNA. Yet during the decade
that this model has dominated, it has not helped to illucidate further the
role of upstream factors.
Prokaryotes (i.e., bacteria) have served as a model for
eukaryotic transcription and it is presumed that the same set of
transcription steps is likely involved in both systems. However,
eukaryotes have many more genes regulated in more complex patterns
and so may require a unique additional level of gene regulation.
Specifically, eukaryotes employ transcription factors that enhance
expression from distances of over a thousand base pairs away from the
initiation site of transcription. Prokaryotes do not have this class of
transcription factors and so do not provide a reliable model for
understanding how eukaryotes regulate transcription.
There is thus a need to identify and characterize the molecular
mechanism of transcription regulation and thereby explain how
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transcription factors work. Once this mechanism is determined, any of a
number of assays for identifying and detecting transcription factors can
be developed. The ability to manipulate the transcription of genes is
necessary to overcorne many obstacles in genetic engineering and gene
therapy technology and will be required to cure several human diseases.
SUMMARY OF THE INVENTION
The invention relates to the discovery that certain transcription
factors catalyze transcription by the creation of single-stranded nicks in
one (and both strands in specialized cases) of the DNA strands of a
gene template. This invention relates to the use of any means of
detection of nicked DNA template or DNA nicking activity to assay
transcription factor activity. This invention further relates to methods of
identifying transcription factors by assaying DNA nicking activity. This
invention further relates to methods of identifying consensus sequences
related to the DNA nicking activity. Furthermore, this invention relates to
the protein domains, and the DNA sequences encoding them, of
transcription factors responsible for DNA nicking activity. This invention
also relates to novel transcription factors identified by the methods of
this invention.
This invention also relates to the use of an assay to detect strand
breakage (nicking) of DNA in response to the binding of one or more
transcription factors (enhancer binding proteins) to determine if these
proteins are actively catalyzing gene transcription. These assays can
be performed on any cell type, or in any cell or tissue in response to any
drug, therapeutic agent or treatment. The DNA nicking assay may be a
gel electrophoresis assay, an SI nuclease assay, a primer extension
reaction, a polymerase chain reaction assay, a protein binding assay or
any combination ther~:of. The analysis of nicking activity may be by
extraction of the tran:;cription factors to assess their activities on
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templates provided in vitro, or endogenous DNA templates can be
extracted and amplified by PCR to determine the in vivo state of
transcription factor induced nicks.
This invention further relates to the adaption of DNA nicking
transcription assays to a 'DNA chip' or any solid or liquid matrix for rapid
screening and analysis of transcription activity. In this matrix screening
technique, double-stranded (ds) DNA oligonucleotides (or ds DNA
fragments containing promoter element, i.e., transcription factor binding
sites) containing DNA consensus sequences capable of specific binding
with transcription factors are fixed to a matrix. The matrix is utilized as a
support to identify specific oligonucleotide sequences, which are
cleaved (nicked) in any solution containing transcription factors.
In the 'DNA chip' screening method of this invention, a DNA chip
containing transcription factor DNA-binding consensus sequences may
be incubated with a soluble cell extract containing representative
transcription factors of any particular cell type in order to assay for
transcription activity. This assay may be used in the evaluation of any
DNA strand containing one or more DNA recognition sequences from a
promoter region, or anywhere else in or surrounding a gene for the
presence of nicks induced by the action of transcription factors. This
method can be utilized as a means of determining which set of
transcription factors and transcription factor binding sites work to
increase gene transcription.
This invention is further directed to a database of transcription
factors that actively nick DNA in any cell type, and in response to any
set of conditions or trE:atrnents. The database may be utilized to predict
which endogenous genes will be actively transcribed in certain cells
based upon DNA sequence information of regions controlling the
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transcription of these genes. In one format of the invention, a useful
database entry wouldl be that a transcription factor, e.g., "XN is
discovered to nick at a DNA binding site in a specific cell type. For
example, fibroblast cells could be analyzed and the DNA sequence that
transcription factors bind to and nick can be rapidly characterized
because the induced nicks are easily detectable. A database of all
transcription factor binding sites controlling each gene is used in
conjunction with the cell type and condition database to realize full
predictive power of the information.
This invention is further directed to a method of identifying
transcription factors and their DNA binding sites utilizing a DNA nicking
assay. In this invention, the database may be utilized to design,
construct and utilize expression vectors capable of efficient expression
in a particular cell type. Once identified, the DNA binding site for the
transcription factor can be engineered into an appropriate expression
vector to induce high levels of gene expression in cells.
This invention is further directed to the use of transcription factors
to create single-stranded nicks in DNA in and around sites dependent
upon the DNA consensus sequence for transcription factor binding.
This use is analogous; to restriction enzymes, which are currently used
to cleave both strand;> of the DNA molecule in, and around the sites of
their specific recognition sequences.
The unique set: of transcription factors of this invention can be
used to specifically cleave DNA. These factors are useful reagents for
molecular biological manipulations.
This invention us further directed to the genetic engineering of
transcription factors in order to alter their ability to nick DNA to yield
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more predictable patterns of behavior. In one format of the invention,
some transcription factors must be phosphorylated on certain amino
acids in order to exhibit DNA nicking activity. For these transcription
factors, replacement. or substitution of potentially phosphorylated amino
acids with acidic residues, such as aspartic acid or glutamic acid, can
allow the transcription factor to actively nick DNA even in the absence of
phosphorylation.
This invention is further directed to the use of genetically
engineered transcription factors for DNA nicking. The engineered
transcription factors of this invention are more reliable than unmodified
transcription factors.
The genetically engineered transcription factors of this invention
may be utilized in vitro as molecular biology reagents or in vivo, by
transformation, transfection or infection, into living cells to alter gene
expression patterns. Cell lines containing genetically engineered
transcription factors may be utilized in conjunction with expression
vectors to produce various therapeutic agents, and similarly employed
for use in gene therapy.
The genetically engineered transcription factors of this invention
may be utilized in a variety of eukaryotic host organisms. Eukaryotes
useful in the invention include fungi, insects, yeast, animals and plant
cells. Although prokaryotes are used for expression of normal or
genetically modified eukaryotic transcription factors, prokaryotes are not
thought to be generally responsive to the class of transcription factors
that induce nicks.
This invention is further directed to the broad use of DNA
template nicking transcription factors to allow RNA polymerase access
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to the transcribed DNA strand by a process of °threading" the
polymerase onto the transcribed strand. It is well known in non-specific
transcription assays that unless eukaryotic RNA polymerase is ofFered a
free DNA end it remains incapable of efficient transcription. The current
paradigm is that GTF~'s substitute entirely for the need of a free end. In
contrast, in this invention a free DNA end is critical to the control of
eukaryotic gene regulation. In this invention, the threading of the
polymerase onto a DNA strand is used as an assay endpoint. Assays
measuring RNA polymerase entry onto the DNA template via a single-
stranded nick, including measurable downstream events, such as RNA
polymerase recognition of the TATA box, or other initiation of
transcription elements or actual initiation of the RNA transcript are within
the scope of this invention.
Presentation of a free DNA end is also a part of the proposed
mechanism, where transcription factors not only nick the DNA template,
but also remain bound to DNA to present a free DNA end to RNA
polymerase. Such a mechanism would explain why a primary property of
many transcription factors that enhance transcription is the presence of
an acidic region, a so~ called acid blob. Positive charges on the 5'
phosphate of a nicked end are repulsed by the positive charges of the
transcription factor, resulting in the deflection of the free end, perhaps
making it more accessible to RNA polymerase.
In this invention, the single-stranded cleavage of one strand of
the DNA double helix by a transcription factor allows access to RNA
potymerase and associated factors and creates an "on" state. In
contrast whereas before the nicking event, RNA polymerase cannot
enter the DNA strand and is in the "off' state. Thus the transcription
factors of this invention can be utilized as 'gene transitors' responsible
for turning on and off genes. The engineering of transcription factor
DNA binding sites to .create arrays of gene transitors for use in logic
operations is also within the scope of this invention.
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BRIEF DESCRIPTION OF THE DRAWINGS
This invention will be better understood by reference to the
drawings, in which:
Figures 1A and 1B are photographs showing autoradiographs of
radioactively labeled gel shift experiments for TFIIIC in nuclear extracts.
Figures 2A and 2B are photographs showing autoradiographs of
radioactively labeled gel shift for TFIIIC.
Figure 3 is a photograph showing an autoradiograph of a
radioactively labeled gel shift experiment.
Figure 4A is a diagram of C-Jun/BPV-E2 fusion proteins. Figure
4B shows the availability of C-Jun/BPY-E2 fusion proteins to bind to
DNA by gel shift assays. Figure 4C shows the relative transcriptional
activity of C-Jun/BPV-E2 fusion proteins.
Figures 5A and 5B are photographs showing autoradiographs of
a radioactively labeled dsDNA oligonucleotide after extraction from a gel
shift experiment.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
To ensure a complete understanding of the invention the
following definitions are provided:
Transcription Factors: Transcription factors are effector
molecules that link second messenger pathways to gene expression
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pathways allowing a range of cellular responses to extracellular stimuli.
Representative transcription factor sequences are available in public
and private databases such as those produced by GenBank and Incyte
Pharmaceuticals and the like. Transcription factors include, but are not
limited to, proteins required for RNA polymerase recognition before
transcription initiation of eukaryotic genes. A recombinant transcription
factor can be a transcription factor encoded by a recombinant DNA
comprising a transcription factor gene sequence or any subsequence
thereof that exhibits transcription factor activity e.g., DNA nicking, DNA
binding, or transcription modulating activity.
DNA template: A DNA template refers to a DNA sequence
comprising a site of direct or indirect interaction for one or more
transcription factors. The DNA sequence may be one occurring
naturally in nature, or may be a recombinant DNA sequence, i.e., one
that is the product of in vitro or in vivo biochemical or genetic
engineering manipulation. DNA isolated from cells, plasmids containing
cloned DNA sequences, and oligonucleotides are all examples of DNA
templates.
Contacting: Contacting means to add to, to mix with, to flow
over, to incubate with" or to co-transfect with, e.g., to introduce two
compounds into a cell. The term also contemplates that the two or more
compounds to be contacted can be co-expressed in vitro or in vivo.
Chlp: A chip, or "biological chip" as used herein, refers to a solid
substrate, for example silicon or glass, having a surface to which one or
more DNA, RNA or protein (peptide) templates are attached. One of
skill in the art will understand that a biological "chip assay" can comprise
the use of chip arrays ar a plurality of biological chips within a single
device or assay.
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D~tection reag~~ent: A "detection reagent" is a compound that can
identify the presence or absence of a nicked DNA strand, e.g., by
binding directly or indirectly to nicks in the DNA. Examples of detection
reagents include, bui: are not limited to, proteins that preferentially bind
to nicks in DNA, such as the X-ray repair cross complementing group 1
protein (XRCC1). Also included are antibodies that bind to proteins that
bind preferentially to nicked DNA.
Detectable label: A detectable label is a compound capable of
being detected and/or measured by, for example, chemical,
biochemical, photochemical, or spectroscopic means. Examples of
detectable labels include radioactive isotopes, enzymes, fluorophores,
chromophores, biotin conjugated antibody, chemiluminescent agents
and the like. Labels may be directly detected through optical or electron
density, radioactive emissions, non-radioactive energy transfers, or
indirectly detected with antibody conjugates, etc.
Nicking transcrietion factor: A nicking transcription factor is a
transcription factor of the present invention, which has the ability to
catalyze a single stranded nick in a double stranded DNA. A
transcription factor of the invention may be a naturally occurring
transcription factor, or a biochemically modified transcription factor, such
as an in vitro phosphorylated nicking transcription factor. Also included
in this definition is a recombinant nicking transcription factor, for
example, a nicking transcription factor modified in vivo or in vitro by
genetic manipulation to contain one or more amino acid substitutions.
Nicked DNA: Nicked DNA is DNA in which one or more of the
phosphodiester bonds linking the various nucleotide bases (adenine,
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guanine, cytosine and thymine) that make up the DNA sequence have
been cleaved or broken.
DNA Nickinc Activi : DNA nicking activity is nucleotide bond
breakage or cleavage in DNA sequences. DNA nicking activity can be
measured by a variei:y of techniques such as (1) changes in
electrophoretic mobility of nicked DNA on polyacrylamide and agarose
gels; (2) determination of nicked DNA by protein binding assays with
proteins that specifically bind nicked DNA; (3) enzyme assays with
enzymes that move progressively along the DNA strand where the
single-strand cleavage of a nick would terminate the reaction, and
permit the identification of the location of the cleavage. Examples of
such enzyme assays are S1 nuclease assays, primer extension
reactions; Polymerase Chain Reaction (PCR) amplification reactions
and DNA sequencing reactions.
In Vivo and In Vitro Nicking Assays: Two categories of
reactions designed to detect the DNA nicking activity are possible. In in
vivo assays, the extent of nicking of endogenous DNA of the cell{s) is
analyzed by methodology similar to that used for in vivo footprinting of
proteins bound to DNA, for example ligation mediated PCR. These
assays determine the state of the DNA as it is acted upon in the living
cell. In in vitro assays, the transcription factors are extracted in their
native states {most similar to their activities in vivo) and are reacted with
DNA in vitro to assess their activity states are measured by nicking
activity.
Taking into account these definitions, the present invention is
directed to methods of analyzing transcription activity and methods of
identifying transcription factors by measuring formation of single
stranded and/or double stranded nicks or breaks in DNA sequence.
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!I. Detection of Nicking Activity
Although transcription factors bind DNA, it is not known that DNA
is altered or nicked by interactions with a transcription factor.
In this invention, as illustrated in Figure 1, a 32P end-labeled DNA
fragment containing the DNA binding site for TFIIIC was incubated with
a protein extract derived from the nuclei of a human fibroblast cell line
(HeLa cells). The 12:9 nucleotide end-labeled VA gene fragment was
incubated in a binding reaction with nuclear extracts prepared from cells
maintained in either IJ.S% or 5.0% serum and infected with either d1312
or Ad2. A nuclear e~;tract from 293 cells was also analyzed. Equivalent
volumes of extract (2N1), derived from equivalent numbers of cells were
assayed in standard binding reactions followed by electrophoresis.
The protein extract contains TFIIIC that is shown to bind to the
DNA as one of two bands shown (arrows). (DNA not complexed with
TFIIIC migrates faster on the polyacrylamide gel and is seen as the dark
bands toward the bottom of the gel.) In cells known to be more
transcriptionally active for pofymerase Ili gene transcription there is
more of an upper band species, whereas cells less active for
polymerase III transcription there is more of the lower band species.
Prior to my invention it was thought that the two bands differed from
each other only in thE~ character of the TFIIIC protein bound. In this
invention I now conclude that the difference between the mobility of the
two bands on the gel is primarily based upon the nicking of the DNA
template in the upper' band species, and that nicking is integral to the
transcription factor (in this case TFIIIC) being able to catalyze
transcription.
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In a second a;~cperiment (Figures 2A and 2B) a 3zP end-labeled
DNA fragment contaiining the DNA binding site for TFIIIC was incubated
with a protein extract: derived from the nuclei of a human fibroblast cell
line (HeLa cells) and the proteins were separated out
chromatographically on a phosphocellulose column.
A HeLa cell nuclear extract (from cells maintained in standard
5.0% serum) was chromatographed on a phosphocellulose column.
Fractions that eluted in the gradient between 0.40 and 0.70 M KCI were
analyzed (Figure 2A) by gel retardation assay using the labeled 129
nucleotide VA, fragment and (Figure 2B) for TFIIIC activity in
reconstituted in vitro transcription reactions using pVA, as the template
DNA. Gel retardation assays were conducted by using 3.5 ~I of each
chromatographic fraction in standard binding reactions. Two series of in
vitro transcription reactions were conducted by using 1.5 ~,I of each
chromatographic fraction and 2.5 ~I of the complementing
phosphocellulose 0.3.5 M step fraction (containing TFIIIB and RNA
polymerase III) derived from cells maintained in 0.5% serum and
infected with d1312. The leftmost lane of each series contains only the
0.35-M step-complementing traction to illustrate that in the absence of
added TFIIIC no VA, transcription is observed.
The chromatography resulted in the separation of 2 forms of
TFIIIC; one form when incubated with the DNA probe forms a lower
band species (arrow ~on left) and the other form appears as the upper
band species (arrow ~on right). In Figure 2, Panel B shows which protein
fractions have transcription activity in an in vitro transcription reaction.
Only the fractions containing the upper band have transcriptional
activity. Prior to my invention, since the TFIIIC proteins that form the
upper and lower bands can be separated chromatographically, it was
thought that only differences in the TFIIIC proteins cause the difference
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in migration of the upper and lower band complexes in the gel. Thus, a
difference in the absolute size of the TFIIIC protein, or its charge,
created the two distinct complexes. With this invention, I now conclude
that the two bands also differ in mobility in the gel because of nicking of
the DNA template. It: thus follows from this experiment that only one
form of TFIIIC possesses the ability to nick the DNA template, and thus
has transcription activity.
In Figure 3, a ~j2P end-labeled DNA fragment containing the DNA
binding site for TFIIIC; was incubated with a single protein fraction
similarly derived as in the previous figure. A protein fraction containing
TFII IC that forms onllr the upper band complex was incubated with the
DNA probe either alone (lane 1 ), or with a phosphatase for increasing
number of minutes (lanes 2-4). The phosphatase enzyme can remove
phosphates on certain amino acids within the TFIIIC protein. Prior to my
invention, it was concluded that the dephosphorylated form of TFIIIC is
smaller (lower molecular weight) than the phosphorylated form of TFIIIC
and so the upper bard is converted to the lower band due to the size of
TFIIIC, and not due to effects on the DNA template. With this invention,
! now conclude that the upper band form is nicked and, therefore, the
dephosphorylated transcription factor can no longer nick the DNA probe,
and so only the lower band forms. Conversely, phosphorylation of the
transcription factor is necessary for it to be active in transcription and to
provide nicking activity.
In Figure 4, a ''2P end-labeled DNA fragment containing the DNA
binding site for BPV-1.2 was incubated with a protein extract where the
cells were transfected with an expression vector that makes a
recombinant transcription factor able to specifically bind this DNA
sequence. This factor c-Jun/BPV-E2 is a hybrid protein containing the
C-terminal part of the BPV-E2 transcription factor (bovine papilioma
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virus E2 DNA binding domain) and the N-terminal part of c-Jun. It
contains the transcriptional activation domain including amino acids
Ser73 (and Ser63) that are phosphorylated in response to signal
transduction (Fig. 4A,). Gel shift assays were conducted using labeled
oligonucleotide containing the DNA-binding site for the BPV-E2 protein.
The oligonucleotides were incubated in whole cell extracts prepared
from cells either not transfected or transiently expressing fusion
proteins. The availability of DNA binding activity for the fusion protein
either containing C-Jun (lane WtE2) or expressing the amino acid
substitutions (lanes T3AE2 and 73DE2) was the greatest for the aspartic
acid substitution (lane 73DE2).
Since the DNA binding site for BPV-E2 is not recognized by any
other transcription fac:,tors in the cells tested, only the transfected fusion
protein c-Jun/BPV-E:? will bind to the 32P-labeled probe (Fig. 4B. no
band visible in the negative control lane 3). Importantly, two bands are
visible if either the normal c-Jun N-terminal end is used to create the
fusion protein (wtE2, lane 2) or if an amino acid substitution of an
alanine for Ser73 is made (73AE2 lane 1, marked with arrows on left of
lanes).
Thus, the phenomenon of two bands observed for TFIIIC in
Figures 1-3 in the claws III system is also observed for a derivative of c-
Jun, in the class II sy:;tem (enhancing catalysis of RNA polymerase II
gene transcription i.e., making messenger RNA). The formation of an
upper band corresponds to increased transcriptional activity as seen
when an aspartic acid residue is substituted for Ser73 (73DE2 lane 4).
In this case the upper' band form predominates (marked with arrow on
right) indicating that an acidic amino acid substitution containing a
negative charge can substitute for the negative charge of a
phosphorylated amino acid in creating an active transcription factor.
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The upper and lower bands are now understood in this invention to differ
by nicking of the DN~4 probe (upper band) when the transcription factor
is activated.
Figure 4C shows the relative transcriptional activity of C-
Jun/BPV-E2 fusion proteins. HeLa, F9, 3T3 and HepG2 cells were
transiently transfected with 2 ug CAT (chloramphenicol
acetyltransferase) reporter gene construct alone or with 2 ~.g of C-Jun or
Jun/BPV-E2 expression vectors. E2CAT, which contains an E2 DNA-
binding site was cotransfected to measure the transcriptional activation
by each of the C-Jun constructs. CAT expression is shown as counts
per minute of benzene-extractable [3H]-mono-acetylated
chloramphenicol per ug extract protein.
Contransfection of a reporter gene construct driven by a promoter
containing a BPV-E2 DNA binding site was used to measure
transcriptional activity of the hybrid protein (Fig. 4C). Whereas an
alanine substitution for Ser 73 had less transcriptional activity (c-
Jun73AE2) than the wildtype (c-JunwtE2) an aspartic acid substitution
(c-Jun73DE2) had greater transcriptional activity in all cell types tested.
These results indicate that genetically engineered transcription
factors can be made that possess either greater or lesser ability to
catalyze transcription, and create DNA nicks, than the normal wildtype
factor. Also, creation of the fusion proteins c-Jun/BPV-E2 illustrates that
a variety of DNA binding domains could be coupled to engineered
transcriptional activation domains to get nicking at a new set of locations
in the DNA sequence as determined by the DNA binding domain.
In Figure 5A a 32P end-labeled DNA fragment containing either no
known DNA binding site (lane 1 ) or the binding site for yet another
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transcription factor, CREB (cyclic AMP-responsive element binding
protein) (lane 2), was incubated with a protein extract derived from the
nuclei of HeLa cells under gel shift conditions, as previously. When
electrophoresed in non-denaturing conditions two bands were observed
to be unique for the CREB binding site. The separation of these two
bands by migration difference in the gel was much greater than
observed for other factors that showed active and inactive forms, and so
these forms might not be related to each other in this way. To
determine how the DNA template is effected by the binding of
transcription factor, the DNA-protein complexes were cut out of the gel.
These gel slices were electroeluted in 1X TAE (0.04Mtris-acetate,
0.001 M EDTA) buffer at 100 volts for 1 hr into a volume of about 200 ~I
in a dialysis bag with a 12,000-14,000 MW cut-off (Spectrum Medical
Industries, Los Angeles). The eluent was made to 0.5% SDS, and 0.1
mg/ml proteinase K .and digested for 15 min. at 56°C. An equal volume
of a 1:1 mixture of phenol/chloroform was added and vortexed and
centrifuged to extract any protein from the labeled DNA. The aqueous
layer containing the DNA template was removed to a fresh tube, and 3
volumes of ethanol were added. A precipitate was allowed to form
overnight at -20°C and the tube was placed on dry ice for 15 min., and
then spun full speed (14,000 rpm) in an eppendorf centrifuge for 15 min
The supernatant was decanted, and the pellet dried. The labeled DNA
pellet was dissolved in standard DNA sample buffer ( 30% glycerol,
0.25% bromophenol blue, 0.25% xylene cyanol), and electrophoresed
on a 15% polyacryla~mide gel running in 1X TBE (0.09 M Tris-borate,
0.001 M EDTA).
Figure 5B shows the labeled template DNA extracted from the gel
shift bands. The template extracted from the bottom band has 2 bands,
as did the input template DNA, but the template extracted from the
upper gel shift band has 3 bands, i.e., an extra band appears. This new
band clearly indicates a change in the template DNA after incubation
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with an extract containing transcription factors. All trace of protein was
removed from the DNA by this procedure, so the new band can only be
formed by a change to the DNA template itself. Two properties of a nick
template would cause it to move more slowly in the gel, as seen in this
figure. Firstly, the breakage of phosphodiester bonds leaves a terminal
phosphate with a positive change. The oligonucleotides used here were
short, about 21 bases pairs of dsDNA. The additional positive charge
would make this fragment move less quickly toward the positive charge
of the electrode in the bottom buffer chamber of the gel tank. Secondly,
nicked DNA has an altered conformation that will allow it to bend at the
location of the nick. 'This change in shape would alter its mobility in a
gel. The changes in the DNA template after binding of transcription
factors is consistent with nicking of the template.
Based upon these results, it is clear that transcriptional activity
can be measured by measuring nicking activity in a host of assays.
III. General Methods of Detecting Transcriptional Activity
DNA nicking activity can be measured by a variety of techniques
including: (1} electrophoresis assays to determine changes in
electrophoretic mobility of nicked DNA on polyacrylamide and agarose
gels; (2) determination of nicked DNA by protein binding assays; (3) SI
nuclease assays; (4) primer extension assays; (5) PCR amplification
reactions (6) DNA sequencing reactions and (7) hypersensitivity to
strand cleavage.
In some of the: DNA nicking assays, oligonucleotide primers are
required to carry out the reaction. Generally, the design and synthesis
of oligonucleotides of the invention follows conventional teachings.
Preferably, oligonucleotides are synthesized on an automated, solid-
phase DNA synthesizer using phosphoramidite chemistry (Beaucage et
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al., 1992; Caruthers, 1983), e.g. Model 392 or 394 DNA synthesizer (PE
Applied Biosysterns, Foster City, Calif.).
1. Electrophoresis Assayrs
Electrophoresis assays can readily be used to identify nicked and
unnicked DNA to assay transcriptional activity.
Molecules of linear double-stranded DNA, which tend to become
oriented in an electric; field in an end-on position, migrate through gel
matrices at rates that are inversely proportional to the logo of the
number of base pairs. Larger molecules migrate more slowly because
of the greater frictional drag and because they worm their way through
the pores of the gels less efficiently than smaller molecules.
A linear DNA fragment of a given size migrates through gels
containing different concentrations of agarose at different rates. Thus,
by using gels of different concentrations, it is possible to resolve a wide
range of DNA molecules.
Superhelical circular (form I), nicked circular (form 11), and linear
(form III) DNAs of the: same molecular weight migrate through agarose
gels at different rates. The relative mobilities of the three forms depend
primarily on the agarose concentration in the gel, but they are also
influenced by the strength of the applied current, the ionic strength of
the buffer, and the density of superhelical twists in the form I DNA.
Under some conditions, form I DNA migrates faster then form III DNA;
under other conditions, the order is reversed.
An unambiguous method for identifying the different
conformational forms of DNA is to carry out electrophoresis in the
presence of increasin~,g quantities of ethidium bromide. As the
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concentration of ethidium bromide increases, more of the dye becomes
bound to the DNA. 'The negative superhelical turns in form I molecules
are progressively removed, the radii of the molecules increase, and their
rate of migration decreases. At the critical free-dye concentration,
where no superhelical turns remain, the rate of migration of form I DNA
reaches its minimums value. As still more ethidium bromide is added,
positive superhelical turns are generated, the DNA molecules become
more compact, and their mobility increases rapidly. Simultaneously, the
mobilities of form li and form III DNA decrease differentially due to
charge neutralization and the greater stiffness imparted to the DNA by
the ethidium bromide'. For most preparations of form I DNA, the critical
concentration of free ethidium bromide is in the range if 0.1 wg/ml to 0.5
~g/ml.
Because plasmid DNA typically has thousands of base pairs, and
the nuclear extracts from cells being analyzed contain many
transcription factors capable of binding at unforeseen locations, analysis
of the conformation of plasmid DNA would work best when incubated
with pure transcription factor preparations. In addition to seeing
differences in plasmid DNA, short DNA fragments of about 100 base
pairs, or less such as oligonucleotides, can migrate differently upon
electrophoresis when nicked as compared to un-nicked DNA. The
phosphodiester strand break creates a free charge, and the DNA can
bend to a greater degree at the location of nicks, causing changes in
migration on gels.
Electrophoresis assays can be readily used to separate and
distinguish nicked from non-nicked DNA and as such, measure
transcription activity and identify transcription factors. Such procedures
are well known in the art as discussed above and as described in
Sambrook, et al. Molecular Cloning A Laboratory Manual 2"d Ed.
CA 02346364 2001-04-06
Ofi-10-2000 PCT/US99/23277
-21-
(1989) Cold Spring Harbor Laboratory Press.
S'~~ 'h f. ~iF
2 .~~" ,'t ;t<fs ~ r~
4. . ~' ~ S .-..r, .....
l' s~t Sf ~ > ... ,
T~ - . . ..
~~~f 5,?;
~
,
.
.
- , ,
i
S
."py
2 , r
06-10-2000 CA 02346364 2001-04-06 PCT/US99/23277
~.
a~a.
D1: WO 90105745 describes the cloning of a CAMP-responsive
transcription enhancer binding protein (CREB).
D2 Cell (1996) 53 907-920 describes the activation of
transcription factor IIIC by the adenovirus E1A protein.
D3 EP-A-O 628 817 describes a method for detecting single-
strand breaks in DNA using monoclonal antibodies directed against
single stranded DNA.
D4 Nucl. Acids Res (1994) 22(7) 1305-1312 describes the
activation of c-Jun transcription factor by substitution of a charged
residue in its N-terminls domain.
D5 FR /A-2 760-025 describes a method for the isolation and/or
assay of damaged DNA, in a sample by binding TBP to a DNA sample.
The method can be used for screening cytotoxic products acting on
DNA.
AMENDED SHEET
CA 02346364 2001-04-06
a i -~
2. Protein g;r,d;n Assayrs
Protein binding asaays can also be used to measure DNA nicking
activity and, therefore, transcriptional activity. Such procedures are well
known in the art as described in Fried, M. and Crothers, D.M. (1981),
Eauilibria and kinetics of Ilac repressor operator interactions by
polyacnylamide qel electrophoresis. Nucl. Acids Res. 9, 6505-6525 and
Garner, M.M. and Revzin, A. (1981 ), A peI electrophoresis method for
guantifving the binding proteins to specific DNA reoions Applications to
the comaonents of the E coli lactose oaeron rectulatory system Nucl.
Acids Res. 9, 6505-3060, each of which is hereby incorporated by
reference. DNA protein binding assays may consist of the minimal
components necessary to permit DNA-protein interaction. The
components of such assays will include a DNA template and a
transcription factor protein (either native or recombinant).
The detection of DNA/protein complex formation involves
determination of an electrophoretic mobility shift of labeled DNA on
:?0 polyacrylamide gels containing, for example, 4% polyacrylamide
(acrylamide:bisacrylamide, 30:0.8), 6.25 mM Tris base, 6.25 mM boric
acid, 0.25 mM EDTA, (equivalent to 114X TBE) electrophoresed at 120
volts (~20 mA at the beginning of the run). To see two bands for the
binding of a single type of transcription factor preelectrophoresis of the
~'.5 gel is important. At a given voltage the amperage of the applied field
increases soon after electrophoresis begins. The amperage needs to
drop back from this spike, back to the starting amperage or slightly
below. Secondly, the TBE buffer used in the gel and during
electrophoresis works better if a 70 X TBE stock solution is made up
30 and allowed to age several months until significant white precipitate
ANIENDED SHEET
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-22-
forms at the bottom of the bottle. Alterations in the formulation of the
TBE buffer to mimic this change should work equivalently, including
altering the ionic strength andlor the pH of the buffer.
Binding reactions can contain, for example, 4-8% glycerol, 0-4%
ficoll, 7 mM MgCl2, 20 rnM Hepes-NaOH pH 7.9, 0.4 mM EDTA, 60-100
mM KCI, 1-4 ~.g poly (dl-dC) poly (dl-dC) in the presence of transcription
factor protein (1-5 pg of either nuclear extract protein or
chromatographic fraction, and DNA. The nuclear extract may be
prepared as described ibelow.
a. Nuclear Extract Preparation
Nuclear extracts are prepared by procedures well known in the
art such as described in Dignam,J.D., Lebovitz, R.M., and Roeder, R.G.
(1983) Accurate transcriation initiation by RNA polymerase II in a
soluble extract from isolated mammalian nuclei. Nucl. Acids Res. 11,
1475-1489. In a modification of this procedure adapted to smaller
numbers of cells growing on tissue culture dishes, cells are removed
from a tissue culture plate (P-100) containing cells with the DNA target
(most cell types including HeLa). The cells are washed twice with PBS
(phosphate buffered saline). Eight hundred microliters of hypotonic
buffer (for example, 10 rnM Hepes pH 7.9, 1.5 mM MgCl2 and 10 mM
KCI) are then added to the cells. The cells are then incubated for 15
min. Next, the cells are scraped off into tubes and 4.8 ~I NP-40 {non-
ionic detergent) is added. The mixture is spun 5 min at 5 G. The
supernatant is aspirated off and the nuclei resuspended in 50 pl lysis
buffer containing 10 mM potassium (or sodium) phosphate pH 8.0 {a
mixture of mono and dibasic mixture to yield pH 8.0), 0.5% NP40, 1.0
mM EDTA (pH 8.0) and 300 mM KCI. The mixture is then incubated for
min at4°C. Next, the' mixture is spun 10 min at 14,000 rpm
30 (Eppendorf centrifuge) at 4° C. The supernatant containing the
nuclear
extract is removed to a fresh tube.
AMENDED SHEET
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Alternative methods for preparing extracts containing soluble
transcription factors also work. For example, the cells can be directly
solubiiized in extraction buffer, without isolating nuclei first, to yield a
whole cell extract.
b. Buffer Composition
Buffer composition is critical for analysis of DNA nicking activity.
Phosphate buffer is preferred for extracting transcription factors in their
active states. Phosphate buffer is preferred because transcription
factors are often phosphorylated and can be dephosphoryiated by
phosphatase activity in the cell extracts if the phosphatase activity is not
inhibited. The phosphate buffer serves as a competitive inhibitor to
endogenous phosphatase. Other biological buffers not containing
phosphate also work for the analysis of nicking activity. The addition of
phosphatase inhibitors, such as sodium flouride at 50 mM, should help
maintain the phosphorylation state of the extracted transcription factors.
c. Detection of Protein-DNA Complexes
The position of the labeled oligonucleotide is detected by
appropriate methods (e.g., autoradiography for radioactive
oligonucleotide). Mobility shifts are evidence of active nicking activity
and thus active transcription activity, particularly when two forms of the
DNA-protein complexes are formed that can be resolved by gel
electrophoresis.
Other methods for detecting or separating DNA-protein
complexes may be used, including UV crosslinking analysis, high
performance liquid chromatography and phage display technology.
Formulation of protein-DNA complexes may be detected as a
retention of labeled DANA (the label being detected by an appropriate
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methodology such as scintillation counting for radiolabeled DNA or
flourometry from fluorescently labeled DNA) utilizing known affinity
methods for protein immobilization. Such methods for protein
immobilization are well known in the art and include biotinlstreptavidin,
nitrocellulose filtratioin, affinity chromatography, immunoaffinity
chromatography, and related techniques.
Protein-DNA complex formation may also be detected as
retention of labeled transcription factor (e.g. radioactively, fluorescently)
utilizing known methods for immobilizing DNA.
3. SI Nuclease Assayrs
Nuclease SI degrades single stranded DNA or RNA to yield 5'-
mono phosphate or oligonucleotides. Double stranded DNA, double
stranded RNA and DNA:RNA hybrids are generally relatively resistant to
the enzyme. However, moderate amounts of the enzyme will cleave
double stranded nucleic acids at nicks or small gaps. As such, SI
nuclease coupled with gel electrophoresis assays can be used to
measure DNA nickinct activity and hence transcription activity.
SI Nuclease assays are well known in the art. One example of
an SI Nuclease assay is as follows. DNA containing a transcription
factor binding site is e:nd labeled and then preincubated with a protein
extract containing the transcription factor activity to be tested. After
nicking has occurred during this preincubation period the DNA is
extracted with phenol/chloroform and ethanol precipitated. The DNA is
subjected to S1 nuclease digestion is at 37°C for 30 minutes in the
presence of 100 ml S-1 mix (250 mM NaCI, 40 mM sodium acetate pH
5.5,1 mM ZnS04, 20 mg/ml denatured salmon sperm DNA and 2000
Ulml S1 nuclease). The reaction is terminated by adding sodium
dodecyl sulfate (SDS;I and EDTA to a final concentration of 0.2% and 20
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mM, respectively, followed by phenollchloroform extraction and
precipitation with 0.3M ammonium acetate and 50% isopropanol. The
precipitate is resuspended in 6 ~,I TE and 4 wl of stop buffer (95%
formamide, 0.05% bromophenol blue, 0.05% xylene cyanol and 20 mM
EDTA) and separated on a fi% polyacrylamide sequencing gel.
Absence of the full-sized band, and the presence of smaller DNA
fragments cleaved by :il, is evidence of nicking activity, as the SI
nuclease has digested the full-length DNA at the position of the nick.
4. Primer E~ctension Reactions
Primer extension reactions can also be used to detect nicking
activity and thereby assay transcriptional activity. The presence of a
nick is required to initiate the primer extension reaction. Detection of a
primer extension product indicates the presence of a nick and, therefore,
transcription activity.
Various primer extension assays are described in U.S. Patents
5,952,202; 5,888,819 and 5,945,284. Primer extension reaction
procedures procedures involve the nicking of the DNA template by a
transcription factor followed by extension reactions. A "primer" refers to
an oligonucleotide capable of selectively annealing to a specified target
nucleic acid and thereafter serving as a point of initiation of a primer
extension reaction. A "primer extension reaction" refers to a reaction
between a target/primer duplex and a nucleotide which results in the
addition of the nucleotide to a 3'-end of the primer such that the added
nucleotide is complementary to the corresponding nucleotide of the
target nucleic acid.
The conditions for the occurrence of the template-dependent,
primer extension reaction can be created, in part, by the presence of a
AMENDED SHEET
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suitable template-dependent enzyme. Some of the suitable template-
dependent enzymes are DNA polymerases. The DNA polymerase can
be of several types. The DNA polymerase must, however, be primer
and template dependent. For example, E. coli DNA polymerase I or the
"Klenow fragment" thereof, T4 DNA polymerase, T7 DNA polymerase
("Sequenase"), T. aquaticus DNA polymerase, or a retroviral reverse
transcriptase can be used. RNA polymerases such as T3 or T7 RNA
polymerase could also be used in some protocols. Depending upon the
polymerase, different conditions must be used, and different
temperatures ranges may be required for the hybridization and
extension reactions.
Nucleic acids of interest may be analyzed by facilitating the
analysis of the 3' terminal addition of terminators to a specific primer or
primers under specific:, hybridization and polymerase chain extension
conditions. Using only the terminator mixture as the nucleoside
triphosphate substrate ensures addition of only one nucleotide residue
to the 3' terminus of the primer in the polymerase reaction. Using all
four terminators simultaneously ensures fidelity, i.e., suppression of
misreading.
By specifically labeling one or more of the terminators, the
sequence of the extended primer can be deduced. In principle, more
than one reaction product can be analyzed per reaction if more than one
terminator is specifically labeled.
By specifically i:agging the oligonucleotide primer(s), or
templates) with a moiety that does not affect the 3' extension reaction
yet permits affinity separation, the extension products) can be
separated post-reaction from the unincorporated terminators, other
components of the reagents, and/or the template strand. Several
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oligonucleotides can be analyzed per extension reaction if more than
one affinity agent is used.
a. Biotinylation of Olgodeoxynucieotides
In some circumstances, it is helpful to biotylate oligonucleotides
to facilitate detection. Oligodeoxynucleotides, terminated at their 5'-
ends with a primary amino group, may be obtained from Midland
Certified Reagents, PJlidland, Tex. These may be biotinylated using
biotin-XX-NHS ester (Clontech Laboratories, Inc., Palo Alto, Calif.), a
derivative of biotin-N~-hydroxysuccinimide. Typically, the oligonucleotide
(9 nanomoles) are dissolved in 100 ~,I of 0.1 M NaHCO/NazC03 (pH 9),
and 25 ~,I of N,N-dimethylformamide containing 2.5 mg biotin-XX-NHS-
ester is added. The mixture is incubated overnight at room temperature.
It is then passed over a 6 ml Sephadex G-25 column ("DNA grade"--
Pharmacia) equilibrated with H20. Eluate fractions containing DNA are
identified by mixing 4 ~,I aliquots with an equal volume of ethidium
bromide (2 p.g/ml) and the DNA-induced fluorescence is monitored with
a UV transilluminator.. Unreacted ester is detected by UV absorption at
220 nm. The tubes containing DNA are pooled, concentrated in a
Centricon-3 microconcentrator (Amicon), and passed over Sephadex
again.
Inhibition of the' binding of 3H-biotin to magnetic M-280
streptavidin Dynabeads (Dynal) can be used to assay quantitatively the
extent of biotinylation of the oligonucleotides. Eppendorf tubes and
pipet tips are siliconiz~ed. A known amount (5-10 pmoles) of biotin-
labeled oligonucleotide in 10 ~,I 0.1 M NaCI is added to tubes containing
25 ~,I of 1:4 suspension of beads in 0.1 M NaCI. Increasing amounts of
3H-biotin (5-35 pmole;>) in 20 pl of 0.1M NaCI are added to the tubes and
these are rotated again for one hour. Tubes are put on a, for example,
Dynal MPC-E magnet to remove the beads from suspension, 10 pl
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aliquots of the supernatant are withdrawn, and the amount of
radioactivity in these' is measured using a Beckman LS 5000 TD liquid
scintillation counter. Counts are compared to those from tubes to which
no oligonucleotide has been added. Alternatively, for some primers,
biotinylation is monil:ored by size fractionation of the reaction products
using analytical polyacrylamide gel electrophoresis in the presence of
8M urea.
b. Prirner Extension/Termination Reactions.
Approximately five pmoles of 5'-biotinylated oligodeoxynucleotide
template are mixed with approximately three pmoles of primer in 1X
sequencing buffer (from Sequenase Version 2.0 kit, US Biochemical
Corp.) (10 p.l final volume). The mixture is incubated at 65°C for
2 min,
then allowed to cool to room temperature in order to anneal the primer
and template. The solution containing the annealed template-primer is
separated into two 5 ~I portions, A and B, to which is added the
following: Reaction ,A (for normalizing template concentrations)--0.5 pl
of 100 mM dithiothreitol, 1 pl each of lOp.M DATP, dGTP, ddCTP, 0.5 pl
of "Mn buffer" (from ;Sequenase Version 2.0 kit, US Biochemical Corp.),
0.5 p.l of 35S-a-thio-d'TTP (10 mCi/ml, 1180 Ci/mmole) (Dupont-NEN), 1
~,I of Sequenase (1:8 dilution, US Biochemical Corp.); Reaction B (for
template-specific labeling of primer 3'-ends)--same additions as in
Reaction A except the nucleotides used were ddCTP, ddGTP, ddTTP,
and 35S-a-thio-ddATl'. Reactions are carried out for 5 min at 37° C.
Control reactions omitting the primer or the Sequenase are also
performed. Aliquots are removed and analyzed by electrophoresis on a
15% polyacrylamide, 8 M urea, DNA sequencing gel (see Maniatis, T.,
et al., Molecular Cloning. a Laboratory Manual, Cold Spring Harbor
Laboratory {1982)). 'The gel is fixed in.10% methanol, 10% acetic acid,
dried down onto Whitman's 3MM paper, and exposed to Kodak X-Omat
AR film. Alternatively, for purposes of analyzing the products by liquid
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scintillation counting" the biotinylated template or template-primer is
bound to an excess of, for example, M-280 streptavidin Dynabeads
(Dynal) before or after the Sequenase reaction (see above,
"Biotinylation of oligodeoxynucleotides", for binding conditions). Beads
are washed three times with 0.1 M NaCI to remove unincorporated label,
then scintillation fluid is added and the radioactivity measured by liquid
scintillation counting.
c. Generation of Templates From PCR Products
Polymerise chain reaction (PCR) reactions are carried out where
one or the other of the amplification primers flanking the target stretch of
DNA are biotinylated as described above. These primers (2 pmol final
concentration) and the target DNA (up to 1 ~,g) are incubated with 2.5
units of Taq polymerise (Perkin EImer/Cetus), 200 pM each of DATP,
dCTP, dGTP, and dTTP, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM
MgCl2, and 0.01 % gelatin (Sigma). Reaction mixtures were overlayed
with paraffin oil and incubated for 30 cycles in Perkin Elmer/Cetus
thermocycler. Each cycle consists of 1 min at 94° C., 2 min at
60° C.,
and 3 min at 72° C. Reaction products are purified by phenollchloroform
extraction and ethanol precipitation, then analyzed by ethidium bromide
staining after electrophoresis on a polyacrylamide gel.
Approximately 5 ~,g of the PCR product is incubated with gentle
agitation for 60 min with 50 ~L of a suspension of prewashed, for
example, M-280 Dynabeads in 0.1 M NaCI. The beads with the bound
DNA (approximately 115 pmoles) are then incubated for 5 min at 25°
C.
with 0.15M NaOH. B~:ads are washed once with 0.15M NaOH to
remove the unbiotinylated DNA strand, then washed three times with
H20. The beads are resuspended in H20 and the strand bound to the
beads via the biotin-streptavidin link is used as template for further
primer extension reactions.
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5. PCR Reactions
PCR reactions can be used to detect DNA nicking activity and
thereby assay transcriptional activity. PCR amplification reactions are
typically conducted in a reaction mixture composed of, per reaction: 1 p,l
genomic DNA containing a promoter from a gene of interest or a
plasmid containing the promoter region; 10 p,l each primer (10 pmol/p,l
stocks); 10 pl 10 x PCR buffer {100 mM Tris.Cl pH8.5, 500 mM KCI, 15
mM MgCl2); 10 ~I 2 mM dNTPs (made from 100 mM dNTP stocks); 2.5
U Taq polymerise (P~erkin Elmer AmpIiTaq.TM., 5 U/pl) or Vent
polymerise at a similar concentration; and H20 to 100 ~I. The cycling
conditions are usually 40 cycles {94° C, 45 sec, 55° C; 30 sec;
72° C, 60
sec) but may need to be varied considerably from sample type to
sample type. These conditions are for 0.2 mL thin wall tubes in a Perkin
Elmer 9600 thermocycler. See, for example, Perkin Elmer 1992/93
catalogue for 9600 cycle time information. The target, primer length and
sequence composition, among other factors, may also affect PCR
parameters.
To detect nick:. using PCR a single-sided PCR reaction can be
conducted and used to map the location of nicks on each strand of the
DNA containing promoter elements. One such protocol is termed
"ligation-mediated PCR", that was developed for genomic sequencing
and direct sequencing reactions, but is adapted in this invention for the
identification and mapping of transcription factor induced nicks. Briefly,
cleaved DNA is denatured and a gene specific primer (from a location
downstream of where the DNA is being tested for the presence of nicks)
is annealed. In first strand synthesis this primer is extended up to the
nicked site creating a blunt site using a processive polymerise, such as
Vent polymerise or Taq polymerise. A staggered linker is attached to
the blunt end by DNA ligase, so that the 5' end of the extended DNA is
06-10-2'000 CA 02346364 2001-04-06 PCT/US99/23277
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ligated to the 3' end of the longer linker strand. The other shorter strand
of the linker lacks a 5' phosphate and is therefore not ligated. A second
primer near the first primer sequence and near the end of the
boundaries of the sequence to be amplified is then annealed. After
addition of a primer to i:he linker region the fragment is amplified by the
polymerase through multiple rounds of PCR, eg. 18 rounds. This
material can be analyzed directly by gel electrophoresis. Alternatively,
the fragment can be radioactively labeled by several methods, including
addition of a labeled 3'~ primer near the second primer that can be
included in additional rounds of PCR to obtain a radioactively labeled
fragment. The fragment is run on a sequencing gel near to a
sequencing ladder created from the same primer to identify the exact
location of the nick.
The use of PCR reactions is unique in that it is adaptable to both
in vivo and in vitro nicking assays. Methods for extraction of genomic
DNA are well known in the art, and can be found in Current Protocols in
Molecular Biology, Supplement 20, 1992. Briefily, in one sample
protocol, cells grown in culture, or present in biopsied tissue are washed
with phosphate buffered saline (PBS). For example, for a 15-cm plate of
cells add1.5 ml of lysis buffer (300 mM NaCI, 50 mM Tris-CI ph 8.0, 25
mM EDTA pH 8.0, 0.2°/. lulu) SDS, and 0.2 mglml proteinase K. Let
plate stand for 5 min. at room temperature (rt). Transfer to 15 ml tube,
and continue incubation at 37°C for 3-5 hr., inverting every 30 min to
mix. Add 1.25 volumes of buffered phenol, mix by inverting about 30
times. Centrifuge to separate phases. Remove aqueous to fresh tube.
Add 1 volume of of by inverting 30 times, and centrifuge to separate
phases. Remove aqueous to fresh tube. Repeat similar extractions with
phenol/chloroformlisoamyl alcohol mix, and then with ethyl ether.
Precipitate DNA with 1 volume of isopropyl alcohol. Pellet DNA,
resuspend in TE, and reprecipitate in
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-32-
ethanol. DNA can now be used for iigation mediated PCR, as described
above
6. DNA Seauencing Reactions
DNA sequencinca reactions can be used to detect nicking activity
and, hence, transcription activity. Methods of DNA sequencing are well
known in the art and include the enzymatic method of Sanger, et al. and
the chemical degradation method of Maxam and Gilbert which are both
described in Sambrook, et al. Other methods of DNA sequencing and
DNA analysis are known and applicable to the present invention. These
methods include the following.
U.S. Pat. No. 5,~~74,527 discloses the use of a low viscosity
medium in capillary elec;trophoretic sequencing of DNA. The preferred
medium is a solution containing between about 4 and about 7 weight
percent linear polyacrylamide molecules. Detection is preferably
performed by detection of fluorescent labels.
U.S. Pat. No. 5,405,746 discloses a method of sequencing DNA
in which the terminus of one strand of a double-stranded DNA molecule
is immobilized on a solid support, for example with a biotin-avidin
system. In the method, the complementary DNA strands are
separated. Next, the unbound strand is removed. Lastly, the
fluorescent- or isotope-labeled Sanger extension products are prepared
on the bound single-stranded DNA molecules. One method for
preparing the immobilized single-stranded DNA is by PCR amplification
using primers with means such as biotin for attaching oligonucleotides,
to produce directly immobilized single-stranded DNA prior to preparing
Sanger extension products. The biotinylated DNA is immobilized on an
AMENDED SHEET
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avidin-agarose gel in a conventional slab format. Radiolabeled reaction
products are detected after electrophoresis.
U.S. Pat. No. 5,484,701 discloses a process for isolating
extension products of PCR amplification in which biotinylated primers
are used in the PCR amplification procedure to produce biotinylated
extension products. In the reaction, the extension products are
immobilized by reaction with a biotinbinding protein such as avidin or
streptavidin. Next, the immobilized products are separated from the
liquid phase of the rE~action and the immobilized complex is denatured
with formamide. Sanger extension products are sequenced by
electrophoresis of radiolabeled or fluorescent products on a gel.
U.S .Pat. No. 5,360,523 discloses a system for sequencing DNA
by electrophoresis on conventional gel slabs or in gel-filled or buffer-
filled capillary tubes, using infrared or near-infrared dyes to label the
bases,. In the method, the bases are labeled with a laser diode to
provide the excitation frequency. An automated scanning microscope
is utilized for detection.
A. Woolley et ~al., "Ultra-High-Speed DNA Sequencing Using
Capillary Electrophoresis Chips," Anal. Chem., vol. 67, pp. 3676-3680
(1995) discloses sequencing DNA by capillary electrophoresis in
polymer-coated capillary channels microfabricated on a glass chip.
Detection is performed by laser-induced fluorescence at visible
wavelengths.
A. Cohen et al., "Separation and Analysis of DNA Sequence
Reaction Products by Capillary Gel Electrophoresis," J.
Chromatogr., vol. 516, pp. 49-60 (1990) discloses the use of
capillary gel electrophoresis to separate DNA sequencing
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reaction products, with detection performed by laser-induced
fluorescence.
Rolfs et al., "Fully-Automated, Nonradioactive Solid-Phase
Sequencing of Genomic DNA Obtained from PCR,"
BioTechniques, vol. 17, pp. 782-787 (1994) discloses binding
biotin-linked DNA to paramagnetic particles coated with
streptavidin in the solid-state for the preparation of purified
Sanger dideouy sequencing ladders with fluorescent dye labeled
primers.
Wlliams et al., "Single-Lane, Single-Fluor
Sequencing using Dideoxy-Labeled, Heavy-Atom Modified Near-
IR Fluorescenlt Dyes," SPIE, vol. 2386, pp. 55-65 discloses the
use of capillary gel electrophoresis in DNA sequencing, in a
polymer-coated silica capillary, with detection by near-infrared
fluorescence. The different ddNTP's are labeled with the same
dye, with molar concentrations varying in a ratio of 4:2:1:0. The
bases are them distinguished from one another by fluorescence
intensity measurements. Also disclosed is the alternative method
of base-calling by measuring fluorescence lifetimes of certain
heavy-atom-modified, near-infrared dyes, with similar absorption
and emission spectra, but different fluorescence lifetimes.
The DNA sequencing methods described in these references
may be employed for the detection of DNA nicking activity and the
analysis of transcription.
7. Hypersensitivit)r to Strand Cleavage
Any of a variety of methods that generate random single stranded
breaks in DNA can ah>o be used to indicate the presence of nicks
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caused by the action of transcription factors. Such methods are, for
example, used in the footprinting of proteins that bind to DNA. Not that
these methods necessarily add much to the nicking induced by
transcription factors, but these methods are appropriately designed to
look at the state of the DNA template, and may indicate specific nicking
along a DNA ladder created by the cleavage reagent. One such reagent
is DNase I that will cut DNA randomly on each strand in the presence of
Mgz' . A footprint results when a DNA binding protein bound to the DNA
template prevents DNase I from cutting. Interestingly, hypersensitive
sites were sometimes noted within or just outside the boundaries of the
protected footprint area, for unexplained reasons. If the transcription
factor bound to DNA is in an active form it can cleave the DNA at a
specific site that may reside within the consensus DNA binding
sequence (or outside of it, as is also the case for restriction enzymes).
Some of these hypersensitive sites may turn out to be nicking sites of
activated transcription factors.
An early technique for indicating promoter region of genes in their
in vivo context was sensitivity of these regions to double stranded
cleavage by DNase. Although the clavage of the DNase is fairly random
along the DNA, regions already nicked would be fully cleaved if
additionally nicked wii:h DNase, resulting in the observed phenomenon
of DNase sensitivity for strand cleavage upstream of active genes.
IV. Use of Chip Technology to Detect Transcription Activity
The above methods of analyzing DNA nicking activity can be
applied and utilized with chip technology.
Cells can be evaluated from any of the body's tissues, or the
tissues of any organism for transcription activity using chip technology.
Cells can be normal, or abnormal, derived from diseased tissue,
including cancer, or before or after drug treatment. Ceils can be isolated
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from any organism including insects, plants, animals, humans, algae,
yeast and fungi. A protein extract of cell nuclei is prepared, as
described above, containing representative transcription factors, as are
present within the nuclei.
A. DNA Chips
A 'DNA chip' containing a population of double stranded DNA
oligonucleotides each containing a potential DNA binding site for a
transcription factor is bound to a known location on the chip. Such chips
are described in U.S. Patent No. 5,837,832. The chip for the present
invention is useful for double stranded DNA of complementary sequence
containing unique sequence motifs that are recognized as binding sites
by transcription factors. The protein extract containing the transcription
factors is incubated with the DNA chip under conditions that allow both
DNA binding and DNA strand cleavage or nicking. In the matrix
screening technique of this invention, double-stranded DNA
oligonucleotides containing DNA consensus sequences capable of
specific binding with transcription factors are fixed to a matrix. The
matrix is utilized as a support to identify specific oligonucleotide
sequences that are cleaved (nicked) in any solution containing
transcription factors.
In the 'DNA chip' screening method of this invention, a DNA chip
containing DNA consensus sequences may be incubated with a soluble
cell extract containing representative transcription factors of any
particular cell type in order to assay for transcription activity. This assay
may be used in the evaluation of any DNA strand containing one or
more DNA recognition sequences from a promoter region, or anywhere
else in or surrounding a gene for the presence of nicks induced by the
action of transcription factors. This method can be utilized as a means
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of determining which set of transcription factors and transcription factor
binding sites work to increase gene transcription.
The methods are particularly suited to automated high throughput
transcription factor screening. In a preferred embodiment, the individual
sample incubation volumes are less than about 500 p,l, preferably less
than about 250 ~l, more preferably less than about 100 ~.I. Such small
sample volumes minimlize the use of often scarce candidate agent,
expensive transcription complex components, and hazardous
radioactive waste.
A variety of methods can be used to create a suitable DNA chip.
For example, a glass slide can be used as a solid support, where the
slide is cleaned and coated with polylysine, and dsDNA is spotted onto
the slide into an array. Alternatively, VLSIPS technology developed by
Affymetrix, Inc. can be used as described in a number of patents such
as U.S. Patent No. 5,8Ei1,242 and 5,945,384. In the VLSIPS method,
light is shone through a mask to activate functional (for oligonucleotides,
typically an -OH) groups protected with a photoremovable protecting
2D group on a surface of a solid support. After light activation, a nucleoside
building block, itself protected with a photoremovable protecting group
(at the 5'--OH), is coupled to the activated areas of the support. The
process can be repeated, using different masks or mask orientations
and building blocks, to prepare very dense arrays of many different
single stranded oligonucleotide probes. A complementary DNA strand
must then be hybridizecl to the affixed ssDNA to yield dsDNA.
New methods for' the combinatorial chemical synthesis of peptide,
polycarbamate, and oligonucleotide arrays have recently been reported
(see Fodor et al., 1991, Science 251: 767-773; Cho et al., 1993,
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Science 261: 1303-13CI5; and Southern et al., 1992, Genomics 13:
1008-10017, each of which is incorporated herein by reference). These
arrays, or biological chips (see Fodor et al., 1993, Nature 364: 555-556,
incorporated herein by reference), harbor specific chemical compounds
at precise locations in ~~ high-density, information rich format, and are a
powerful tool for the study of biological recognition processes that could
be utilized in the present application.
The DNA chips, prepared in any of a variety of these ways, are
then incubated with nuclear extracts containing soluble transcription
factors prepared from a broad sampling of cell types and tissues from a
variety of eukaryotic organisms. In each case, the inclusion of a
phosphatase inhibitor is important to maintaining transcription factor
activity levels in the extracts, and can be provided by use of a buffer
75 containing phosphate, as described above. The nuclear extracts are
incubated with the DNA chips for several minutes to hours at 30°C to
37°C in order to allow cleavage of DNA strands by active transcription
factors.
Detection of the DNA nicks can be accomplished by a variety of
methods, and fall into four classes: proteins that bind to nicks, filling in
of
the nick with a detectab~ie tag, detecting a stalled enzyme at the position
of the nick, removal of a portion of the DNA strand separated from the
rest of the strand by the nick achieved by denaturation.
a. Proteins (or Chemicals) That Bind To Nicks
A number of different proteins are capable of binding to nicks,
and can be used to detect nicks. For example, several proteins involved
in DNA repair will recognize and bind to nicks in DNA. These proteins
(or chemicals) can be themselves coupled to a detector molecule, such
as a radioactive tag, a colormetric tag, a fluorescent or phosphorescent
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tag, or they could be detected by a second molecule that will recognize
them, such as an antibody. Binding candidates include all or part of the
complex that recogniize nicks in eukaryotic cells, that includes the X-ray
repair cross complementation group 1 protein (XRCC1), DNA ligase III,
DNA polymerase , and PARP (Poly ADP Ribose Polymerase). XRCC1
has been shown to bind to nicked DNA itself, a property maintained by
the N-terminal domain (NTD). Thus XRCC1, or XRCC1 NTD could be
used to detect transcription factor induced nicks.
PARP was once thought to be a general transcription factor
(TFIIC) because of it;s requirement in in vitro transcription reactions if the
template DNA contained nicks, and TFIIH that couples transcription to
DNA repair. TFIIH has multiple subunits and any of these individually
may be appropriate including XPB, XPD and p62. Any proteins found to
have high affinity for nicks can be used, such as helicases,
topoisomerases, DNA ligases, or DNA polymerases. Potentially, single-
stranded DNA binding proteins can also bind selectively to nicks, as are
available from eukaryotes, prokaryotes, and their viruses or
bacteriophages, respectively. The HU protein from E.coli has a KD of
8nM for a 30-mer duplex DNA containing a nick, and so can also be
used as a detector protein for transcription factor induced nicks.
The presence of the nick binding protein to a spot on the DNA
chip can be detected in a variety of ways. The protein itself can be
labeled with a radioactive tag, a colormetric tag, a fluorescent or
phosphorescent tag. The protein can also detected by a secondary
protein such as an antibody specific for the protein, or an antibody
specific for a tag that is engineered to be contained in the nick detector
protein. The antibody can contain the radioactive tag, a colormetric tag,
a fluorescent or phosphorescent tag. Alternatively, a secondary
antibody recognizing the first antibody could have these tags.
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b. Fillings In Nicks With A Detectable Tao
Several of the proteins mentioned above possess enzymatic
activity for repair of the nicked DNA. For example, PARP will repair
nicks in DNA by insertion of the terminal phosphate of ATP to reconnect
the phosphodiester backbone. The inserted phosphate can be labeled
to be detectable, by radioisotope tagging or by some other chemical
modification. Likewi:ce the nick may be repaired by TFIIH, a DNA
polymerase or a DNA ligase, and in each case the repair reaction can
insert a tagged molecule that can be detected as a positive signal in our
assay.
Reaction intermediates can also be used to label the nicks. For
example, DNA ligase incubated in the presence of nicked DNA and ATP
will form a DNA-AMP complex at 0° C, observed to be more stable at pH
6.5 than at 7Ø A labeled group on the AMP can then be used to score
the presence of the nick.
c. Detecting a Stalled Enzyme At The Position Of Nicks
Enzymes that processively move along DNA will typically stall at
the strand break of a nick, and this can be used to detect the nick itself.
For example, DNA polymerase will stall at a nick during DNA synthesis
because it cannot traverse the break in the phosphodiester backbone. A
second detector molecule could detect the presence of the enzyme.
Conditions can be used where only the stalled enzyme would remain
bond to the template, such as a high enough ionic strength to dissociate
an enzyme that is nol: stalled, while still allowing stalled enzymes to
remain bound. Competitor DNA could also be a titrated ingredient of the
wash solution for the detector DNA chip, allowing stalled enzymes to
remain bound while other enzymes would be likely to become bound to
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competitor DNA, and so could be washed away thereby allowing
detection of stalled enzymes.
d. Strand SelJaration Of Unattached DNA Fragment Created By_
Nicks
If at least one of the strands of the dsDNA fragment containing
the transcription factor binding site is attached to the solid support of the
DNA chip then part of that strand will remain bound even after nicking of
that strand has occurred. This opens up the opportunity to cleave off a
detector molecule attached to the other side of the nick that would be no
longer attached to the solid matrix. Heating, or other method of
denaturation could be used to separate the two strands of the dsDNA,
thereby allowing the fragment to be washed away. Likewise, if the non-
nicked strand were aNso attached to the solid support then once the free
fragment from the nicked strand is washed away a second labeled
probe can be used to detect the unpaired strand, thereby detecting a
nicked strand.
B Robotic Equipment
The methods of this invention are well suited for automation,
especially computerized automation. Accordingly, a computer-
controlled electromechanical robot preferably performs the method
steps. While individual steps may be separately automated, a preferred
embodiment provide; a single computer-controlled multifunction robot
with a single arm axially rotating to and from a plurality of work stations
performing the mixture forming, incubating and separating steps. The
computer is loaded with software which provides the instructions which
direct the arm and work station operations and provides input (e.g.,
keyboard and/or mouse) and display (e.g., monitor) means for operator
interfacing.
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In a particular embodiment, the robotic station comprises a
robotic arm with axially-positioned work stations including a working
source plate station, a working pipette tip station, a working assay plate
station, a liquid dispensing station, a wash station, a multiple channel
pipettor station, a shaker station, a cooling station and a pipette tip
storage station. In one format of the invention, the arm retrieves and
transfers a microtiter plate or biological chip to a liquid dispensing
station where measured aliquots of an incubation buffer and a solution
comprising one or more candidate agents are deposited into each
designated well. The; arm then retrieves and transfers to and deposits in
designated wells a measured aliquot of a solution comprising a labeled
transcription factor protein. After a first incubation period, the liquid
dispensing station deposits in each designated well a measured aliquot
of a nucleic acid solution. The first and/or following second incubation
may optionally occur after the arm transfers the plate to a shaker station.
After a second incubation period, the arm transfers the microtiter plate to
a wash station where the unbound contents of each well is aspirated
and then the well repeatedly filled with a wash buffer and aspirated.
Where the bound label is radioactive phosphorous, the arm retrieves
and transfers the plate to the liquid dispensing station where a
measured aliquot of scintillation cocktail is deposited in each designated
well. Thereafter, the mount of label retained in each designated well is
quantified.
DNA chips containing arrays of oligonucleotide probes can be
used to determine whether a protein extract has DNA nicking activity.
The array of probes comprises probes exactly complementary to the
reference sequence, .as well as probes that differ by one or more bases
from the exactly complementary probes.
C. Database of Transcription Factors
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This invention is further directed to a database of transcription
factors that actively nick DNA in any cell type, and in response to any
set of conditions or treatments. The database may be utilized to predict
which genes will be actively transcribed in certain cells based upon DNA
sequence information of regions controlling the transcription of these
genes. In this invention, the database may be utilized to design,
construct and utilize expression vectors capable of efficient expression
in a particular cell type. In one format of the invention, a useful
database entry would be that a transcription factor, e.g., "X" is
discovered to nick at a DNA binding site in a specifc cell type, for
example fibroblast cells and the DNA sequence that it binds to and nicks
can be characterizedl.
IV. DNA Nicking Activity Consensus Sequences
One of several methods can be used to determine which of the
DNA oligonucleotides are cleaved. For example, a variety of proteins,
or other molecules, acre known to preferentially bind to nicks in the DNA,
such as TFIIH or a single-stranded DNA binding protein. These proteins
can be fluorescently tagged, or marked to facilitate detection. The chip
is then 'read' to determine which DNA sequences were specifically
nicked by active transcription factors. Since each transcription factor
preferentially binds to only certain DNA consensus sequences, an
inference is made between the sequence of the DNA oligonucleotide
bound to the chip that was cleaved, and possible transcription factors
known to bind to such a consensus sequence. Currently DNA chips are
used to determine the presence of an RNA transcript, or as a means of
DNA sequencing, based on hybridization between DNA strands. Here,
DNA chips find a completely new purpose in assessing the set of
transcription factors in active states, ready to catalyze transcription, in
any given cell type from any organism type.
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Any intact promoter region of any gene, or any region containing
a binding site for a transcription factor can be assessed to determine
when the factors are actively catalyzing transcription. The use of larger
intact contiguous DNA strands may allow an assessment of which
factors are working to activate a particular gene. Unlike DNA
oligonucleotides that may be only large enough to contain one or two
binding sites for transcription factors, a larger strand of DNA may allow a
larger number of transcription factors to bind. The interaction of several
factors together may also cooperate to create a nick on a gene to
catalyze transcription. In this case more traditional methods of detecting
the nick could be used, including S1 nuclease, primer extension
reactions, PCR, or DNA sequencing reactions as discussed above.
V. Methods of Modulating Transcriptional Activity
Some transcription factors must be phosphorylated on certain
amino acids in order to exhibit DNA nicking activity. For these
transcription factors, replacement or substitution of potentially
phosphorylated amino acids with acidic residues, such as aspartic acid
or glutamic acid, can sallow the transcription factor to actively nick DNA
even in the absence of phosphorylation. Conversely, the substitution of
an amino acid that cannot be phosphorylated, such as alanine would
inactivate the transcription factor.
Amino acid sites within transcription factors that are
phosphorylated in response to signal transduction are candidate sites
for the control of transcriptional activity and associated DNA nicking
activity. If the transcription factors have already been cloned, these
sites can be mapped by incubating living cells with 32P (inorganic
phosphate) while stimulating a certain signal transduction pathway that
ends in the activation of a transcription factor. The factor will
incorporate 32P at amino acids (Ser, Thr, Tyr) that may need to be
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phosphorylated in order for the transcription factor to be activated. The
location of the phosphorylated amino acid can be determined by
mapping the 32P labeled amino acid of a tryptic (or other protease)
fragment, as typically resolved on either thin layer chromatography
plates or on HPLC columns.
The amino acids that are phosphorylated in response to signal
transduction are candidates for controlling transcriptional activation and
thus DNA nicking activity. Acidic residues, either Asp or Glu, are
genetically engineered to substitute for the amino acid substrates of
phosphorylation. The thus modified transcription factors are tested for
their ability to nick DNA. Overexpression of such engineered
transcription factors could provide a whole new set of useful molecular
biology reagents, creating enzymes that can effect single stranded
cleavage of DNA at precise locations determined by the DNA sequence,
much like restriction enzymes that are currently used to cut both strands
of the DNA.
VI. Kits
The invention also provides kits useful for performing screens for
activated transcription factors. Also provided are kits useful for assaying
the level of transcriptional activity in an extract, cell or tissue. Kits of
the
invention comprise sl:andardized reagents used to perform standardized
methods for screening and measuring transcription by detecting DNA
nicking activity. Kits may include, inter alia, containers or matrices for
performing transcription assays, DNA templates for transcription factor
binding or cleavage, suitable buffers for DNA binding and cleavage,
reagents useful for detecting nicks in a DNA molecule, and written
instructions.
VII. Biological Switches and Logic Operators
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The identification of nicking activity as the key to transcription
activity can be used as a biological switch. An analogy can be made
between transcription factors of this invention and electronic transitors.
A transitor is an electronic switch that controls the flow of electrons in a
circuit. Similarly, these transcription factors of this invention function as
'gene transitors' controlling the flow of the enzymes (e.g. RNA
polymerase) that lead to the expression of a gene. The invention
therefore includes any use made of this class of transcription factors
and their DNA consensus sequences as switching mechanisms, as
constructed naturally, or in artificial assemblies. There are either of two
positions, either the DNA is nicked or it is not, giving rise to the
rudimentary element of computer logic. The definition of whether the
switch is on or off can be determined by the state of the flow of
molecules along the DNA. If RNA polymerase, another GTF, or any
processive molecule able to travel down a DNA strand gains access to
DNA through the nick then the nick is equivalent to being in the 'on'
position. DNA repair enzymes can be made to reseal nicked DNA, so
the switched can be l:urned off.
A subsequent potentially nicked region can be placed
downstream of the first and present a potential barrier to the movement
of the polymerase down the strand. Similar to two switches in series,
the state of this biological logic gate is determined by whether the DNA
is nicked at either location. Although one of the positions needs to be
nicked to allow polymerase entry, nicking of the downstream site can
create a potential barrier to the polymerase. In this case the definition of
'on' and 'oft' changes, so that a second downstream site must kept
whole to be on, and is turned off by a nick. Transcription factor binding
sites can be arranged into logic assemblies by adjacent placement on a
DNA strand. Use of these assemblies can be as computers, or other
nanomachines.
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VI1. Relevant; Prior Art
In addition to the: prior art references discussed above, relevant
patents, patent applications and publications include the following. U.S.
5,143,854; WO 90115070; W092110092, Hoeffier, W.K., Kovelman,R.,
and Roeder, R.G. (1988). Activation of Transcription Factor IIIC by the
Adenovirus E1A Proteiin. Cell 41, 955-963. Hoeffler, W.K., Levinson,
A.D., and Bauer, E.A. (1994). Activation of cJun transcription factor by
substitution of a charged residue in its N-terminal domain. Nucl. Acids
Res. 22, 1305-1312. Marintchev, A., Mullen, M.A., Maciejewski, M.W.,
Pan,B., Gryk, M.R., and Mullen, G.P. (1999) Solution structure of the
single-strand break repair protein XRCC1 N-terminal domain. Nat Struct
Biol 6(9), 884-893.
The invention now being fully described it will be apparent to one
of ordinary skill in the art that many changes and modifications can be
made thereto without departing from the spirit or scope of the appended
claims.
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