Compositions and methods for characterizing a DNA repair variant polypeptide
View Patent ↗As described below, the present invention provides quantitative homologous recombination assays developed to characterize the pathogenicity DNA repair polypeptides (e.g., BRCA1, BRCA2, Rad51) and provide urgently needed functional information on the significance of DNA repair variants of uncertain significance (VUS) alleles. The invention also provides a method of generating site-specific recombination at a genomic locus or site-specific genome editing by inhibiting replication at the genomic locus, e.g., involving contacting the genomic locus with polypeptides that specifically bind target sequences at the genomic locus.
1. A method of treating breast cancer in a selected patient the method comprising administering a PARP inhibitor or cisplatin to the patient, wherein the patient is selected by a method comprising:
(a) sequencing a DNA repair gene in a biological sample derived from the patient, thereby identifying a mutation in the DNA repair gene;
(b) contacting a mammalian cell that lacks a DNA repair polypeptide with each of: a vector encoding a DNA repair polypeptide comprising the identified mutation and a vector encoding a wild-type Tus polypeptide fused to a nuclear localization signal, wherein the mammalian cell comprises
(i) a single copy of a polynucleotide comprising a 5′ truncated GFP encoding sequence positioned upstream of 5′ and 3′ RFP exons positioned upstream of one to six Ter sites positioned within a GFP encoding sequence comprising a rare cutting endonuclease site, wherein the polynucleotide is integrated into the cell genome; and
(ii) an expression vector encoding a wild-type Tus polypeptide: and
(c) detecting long-tract gene conversion in the cell, wherein an increase in long tract gene conversion in the cell relative to a reference cell expressing a wild-type DNA repair polypeptide indicates that the mutation in the DNA repair polypeptide is functionally significant; and
(d) selecting the patient having a functionally significant mutation in the DNA repair polypeptide.
2. The method of claim 1 , wherein the DNA repair polypeptide is one or more of BRCA1, BRCA2, and Rad5.1.
3. A method of treating breast cancer in a selected patient, the method comprising administering a PARP inhibitor or cisplatin to the patient, wherein the patient is selected by a method comprising:
(a) sequencing a DNA repair gene in a biological sample derived from the patient, thereby identifying a mutation in the DNA repair gene;
(b) contacting a mammalian cell that lacks a DNA repair polypeptide with each of: a vector encoding a DNA repair polypeptide comprising the identified mutation and a vector encoding a wild-type Tus polypeptide fused to a nuclear localization signal, wherein the mammalian cell comprises
(i) a single copy of a polynucleotide comprising a 5′ sequence encoding a truncated first reporter polypeptide positioned upstream of 5′ and 3′ exons encoding a second reporter polypeptide positioned upstream of one to six Ter sites positioned within polynucleotide is integrated into the cell genome; and
(ii) an expression vector encoding a wild-type Tus polypeptide; and (c) detecting long-tract gene conversion in the cell, wherein an increase in long tract gene conversion in the cell relative to a reference cell expressing a wild-type DNA repair polypeptide indicates that the mutation in the DNA repair polypeptide is functionally significant; and
(d) selecting the patient having a functionally significant mutation in the DNA repair polypeptide.