DNA structure specific recognition protein complexes
AssigneeMassachusetts Institute of Technology
InventorsBrian A. Donahue, Jeffrey H. Toney, John M. Essigmann, Stephen J. Lippard, Pieter M. Pil, Suzanne L. Bruhn, Steven J. Brown, Patti J. Kellett
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Abstract
DNA structure specific recognition protein of eukaryotic origin and DNA encoding such a factor, as well as probes specific for DNA structure specific recognition protein or DNA encoding it and methods of detecting DNA structure specific recognition protein in eukaryotic cells. In particular, a mammalian cellular factor that selectively recognizes and binds DNA damaged or modified by a drug (the anticancer drug, cis-diamminedichloroplatinum (II) or cisplatin) has been identified.
Claims19
A (damaged DNA fragment):(protein) complex comprising a DNA structure specific recognition protein bound selectively to a double-stranded DNA fragment having at least one region of DNA damage comprising a 1,2-intrastrand d(ApG) or d(GpG) dinucleotide adduct, said complex having a retarded electrophoretic mobility relative to the mobility of the damaged DNA fragment alone.
A (damaged DNA fragment):(affixed protein) complex comprising a DNA structure specific recognition protein affixed to a solid support, said affixed protein being bound selectively to a double-stranded DNA fragment having at least one region of DNA damage comprising a 1,2-intrastrand d(ApG) or d(GpG) dinucleotide adduct.
An isolated eukaryotic DNA structure specific recognition protein having the properties of:
- (a) selective binding to a double-stranded DNA fragment having at least one region of DNA damage comprising a 1,2-intrastrand dinucleotide adduct, including the 1,2-d(ApG) and 1,2-d(GpG) diadducts of cisplatin, to form a (damaged DNA fragment):(protein) complex which has a retarded electrophoretic mobility relative to the mobility of the damaged DNA fragment alone;
- (b) when affixed to a solid support, said protein binds selectively to said double-stranded DNA fragment having at least one region of DNA damage comprising a 1,2-intrastrand dinucleotide adduct, and not to a double-stranded DNA fragment lacking said region of DNA damage;
- (c) sedimentation through a sucrose gradient with a coefficient of about 5.6S;
- and
- (d) SDS-PAGE electrophoretic mobility corresponding to an apparent molecular weight of about 91,000 daltons.
A recombinant protein having DNA structure specific recognition activity, encoded by a nucleotide sequence selected from the group consisting of:
- (a) SEQ ID No. 6;
- (b) a nucleic acid sequence that specifically hybridizes to SEQ ID No. 6;
- (c) SEQ ID No. 8;
- (d) SEQ ID No. 9;
- (e) a nucleic acid sequence that specifically hybridizes to SEQ ID No. 8;
- (f) a nucleic acid sequence that specifically hybridizes to SEQ ID No. 9;
- and
- (g) the .lambda.Pt1 gene present in E coli recombinant cells, ATCC Deposit No. 40498.
A recombinant protein having DNA structure specific recognition activity, comprising the amino acid sequence of SEQ ID No. 7.
A recombinant protein having DNA structure specific recognition activity, comprising the amino acid sequence of the HMG-box region of SEQ ID No. 7.
A recombinant protein having DNA structure specific recognition activity, encoded by a nucleic acid sequence selected from the group consisting of:
- (a) SEQ ID No. 10;
- and
- (b) a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of SEQ ID No. 10.
A recombinant protein having DNA structure specific recognition activity, comprising the amino acid sequence of SEQ ID No. 11.
A recombinant protein having DNA structure specific recognition activity, comprising the amino acid sequence of the HMG-box domain region of SEQ ID No. 11.
A recombinant protein having DNA structure specific recognition activity, a substantial portion of which is encoded a nucleotide sequence selected from the group consisting of:
- (a) the .lambda.yPt gene;
- (b) the nucleotide sequence of SEQ ID No. 12;
- and
- (c) a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of SEQ ID No. 12.
A recombinant protein having DNA structure specific recognition activity, comprising the amino acid sequence of SEQ ID No. 13.
A recombinant protein having DNA structure specific recognition activity, comprising the amino acid sequence of an HMG-box domain region of SEQ ID No. 13.
Dependencies are inferred from the claim text.