Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-04
2001-11-06
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300
Reexamination Certificate
active
06312897
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the study of protein-DNA interactions, and more particularly to a technique for inhibiting protein-adenine noncovalent contacts without disrupting the secondary structure of DNA, thereby providing a template-directed interference technique for precisely studying protein-DNA interactions.
BACKGROUND OF THE INVENTION
The study of molecular recognition at the interface between protein and DNA is of paramount importance in the study of protein regulation of gene expression. Such protein-DNA recognition involves noncovalent interaction (hydrogen bonding, ionic interactions, and nonpolar interactions) between nitrogen bases of DNA and amino acids of protein characterized by great stability and specificity.
A variety of techniques, referred to as DNA footprinting, have been developed to study these interactions. These techniques typically involve chemical alteration of DNA to inhibit interaction with protein, exposure of the DNA to protein, separation of protein-bound DNA from unbound DNA, and subsequent DNA cleavage and sequencing, or exposure of DNA to protein followed by addition of a chemical moiety that will interact with DNA except where protected by protein, followed by cleavage and sequencing. The results of a polyacrylamide sequencing gel can identify the location of protein binding in these procedures via identification of a lack of DNA fragments in a molecular weight region that would have been present without protein binding at a specific location, or presence of bands signifying DNA fragments that are present because DNA was not allowed to bind at a specific location.
Specifically, in “DNA protection footprinting”, protein is bound to end-labeled DNA and the resulting complex is treated with a reagent that interacts with DNA (but not at regions bound to protein) to produce chemical lesions that give rise to strand cleavage. The conditions of treatment are adjusted such that each DNA molecule receives statistically one lesion, thereby producing a statistical mixture of singly-modified DNA molecules each including a cleavage site at a random position along the DNA chain, but not at the site of protein binding. Subsequent cleavage and gel sequencing of the cleaved, protein-bound DNA, and comparison with a sequenced control of DNA treated identically but without protein binding, results in a lack of bands associated with protein binding positions.
“DNA interference footprinting” involves treatment of end-labeled DNA with a reagent that prevents protein binding and provides a cleavage site, to produce a probe of statistically singly-modified DNA molecules. The pool of DNA is incubated with DNA-binding protein, protein-bound DNA is separated from unbound DNA, and both populations are subjected to cleavage conditions. The resulting fragments are separated on a sequencing gel and, by comparison of the bands representing bound DNA with bands representing unbound DNA, a lack of bands in the protein-bound fraction and presence of bands in the protein-unbound fraction, each in a region corresponding to a molecular weight distribution of fragmentation at a particular location on the DNA, is indicative of protein binding at that region.
Other footprinting techniques, such as DNase I footprinting, Exonuclease III footprinting, hydroxyl radical footprinting, diethyl pyrocarbonate footprinting, KMnO
4
and OsO
4
footprinting, ethylation interference footprinting, uranyl photofootprinting, methylation protection and methylation interference footprinting, and missing contact footprinting are known and are described by C. J. Larson and G. L. Verdine, “The Chemistry of Protein-DNA Interactions”,
Bioorganic Chemistry: Nucleic Acids
, S. M. Hecht, Ed., Oxford University Press, New York, 1996, pages 324-342. Most of the above techniques do not provide information as to which groove of DNA, major or minor, is involved in binding.
An optimal footprinting method for determining specific base contacts at Protein-DNA binding sites should possess a number of characteristics, including the following: (1) the method should be capable of assaying contacts to all four bases in both the major groove and the minor groove of DNA, (2) the structure of the interfering probe moiety should be known, (3) the interference probe should minimally perturb DNA secondary structure relative to its natural counterpart, and (4) the method should be operationally simple. Unfortunately, many of the footprinting methods described above fall short of these goals since they involve attachment of a chemical moiety to DNA that can affect larger-scale phenomena such as DNA conformation.
Accordingly, template-directed interference (TDI) footprinting (described also in the above-reference article of Larson and Verdine) has been developed to circumvent the chemical reagent-based approach. TDI footprinting relies upon the ability to incorporate, in the DNA polymer itself, a molecule that is similar enough to one of the nucleic acids to avoid disruption of DNA secondary structure, that possesses the ability to base-pair as would a normal nucleotide, but that disrupts protein-DNA interaction, and is cleavable. TDI footprinting offers significant advantage over others of the above-described footprinting methods in that alteration of DNA secondary structure is avoided, and DNA and/or protein is not induced to act in a manner inconsistent with natural protein/DNA bonding.
Although TDI footprinting offers significant advantage in the study of protein-DNA interaction, availability of the necessary nitrogen base analogs for the technique is limited because of the challenging requirements discussed above. TDI footprinting analogs of guanine, cytosine, and thymine have been reported (Hayashibara, K. C., Verdine, G. L.,
J. Am. Chem, Soc.,
1991, 113, 5104-5106; Hayashibara, K. C., Verdine, G. L.,
Biochemistry,
1992, 31, 11265-11273; Mascareñas, J. L., Hayashibara, K. C., Verdine, G. L.,
J. Am. Chem. Soc.,
1993, 115, 373-374, respectively). However, although TDI footprinting has been known since at least 1991, and cited extensively, TDI footprinting of the DNA base adenine has remained elusive. Significantly, without an available, suitable TDI footprinting analog of adenine, TDI footprinting as a technique has been constrained in that it could not be used to analyze contacts to the base surface of the entire major groove of DNA, the principal locus of sequence-specific interactions in protein-DNA complexes.
It is, therefore, an object of the present invention to provide TDI footprinting involving adenine.
SUMMARY OF THE INVENTION
The present invention provides a molecule that mimics adenine, in that it can be incorporated into DNA without adversely affecting normal base pairing to thymine, but that disrupts adenine-dependant DNA binding to proteins. The molecule find particular utility in a method also provided by the invention that involves study of protein-DNA interaction.
The molecule of the invention has a formula:
where X is a moiety allowing cleavage of double bond 12 under conditions tolerable by a protein-DNA complex, R is H or a hydrocarbon, and Z is an oligonucleotide, 2′-deoxyribose-5-′triphosphate, or a functionality attachable to an oligonucleotide (which can be selected by one of ordinary skill in the art). According to another aspect of the invention, an oligonucleotide, such as DNA, including at least one nitrogen base of the above formula is provided.
A method of the invention involves a technique for determining contacts in a protein-DNA complex. A plurality of end-labeled DNA strands are provided, each including, statistically, a nitrogen base of the above formula. A DNA-binding protein is added to the plurality of strands, protein-bound DNA is separated from unbound DNA, and the DNA strands are cleaved. The strands then are sequenced to determine a molecular weight region populated predominantly by the unbound strands, representative of cleavage sites indicating a protein-binding site.
The invention also provides a method of determining co
Cushing Timothy D.
Verdine Gregory L.
Houtteman Scott W.
President and Fellows of Harvard College
Wolf Greenfield & Sacks P.C.
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