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 characterizing the functional significance of a mutation in a DNA repair polypeptide, the method comprising:
(a) expressing in a mammalian cell a DNA repair polypeptide comprising a mutation and a wild-type Tus polypeptide fused to a nuclear localization signal, wherein the cell comprises a single genomic integrated copy of a polynucleotide comprising, from 5′ to 3′: a promoter directing expression in the mammalian cell, a 5′ truncated green fluorescent protein (GFP)-encoding sequence, a 3′ red fluorescent protein (RFP) exon, a 5′ RFP exon, and mutated GFP encoding sequence comprising one to six Ter sites adjacent to a rare cutting endonuclease site; and
(b) detecting long-tract gene conversion in the cell, wherein an increase in long tract gene conversion in the cell relative to a reference indicates that the mutation in the DNA repair polypeptide is functionally significant.
2. The method of claim 1 , wherein the DNA repair polypeptide is BRCA1, BRCA2, BARD1, PALB2, RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, BLM, RecQ helicase, MRE11, Rad50, NBS1, ATM, ATR, CTIP, Brip, RPA, RPA-like polypeptide, and combinations thereof.
3. A method of characterizing the functional significance of a mutation in a DNA repair polypeptide in a biological sample, the method comprising
(a) sequencing a DNA repair gene in a biological sample derived from a subject, thereby identifying a mutation in the DNA repair gene;
(b) contacting a mammalian cell lacking a DNA repair polypeptide and comprising a single genomic integrated copy of a polynucleotide comprising, from 5′ to 3′, a promoter directing expression in the mammalian cell, a 5′ truncated green fluorescent protein (GFP)-encoding sequence, a 3′ red fluorescent protein (RFP) exon, a 5′ RFP exon, and a mutated GFP encoding sequence comprising one to six Ter sites adjacent to a rare cutting endonuclease site, with each of:
a vector encoding a DNA repair polypeptide comprising the identified mutation of step (a), and a vector encoding a wild-type Tus polypeptide fused to a nuclear localization signal; 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.
4. The method of claim 3 , wherein the DNA repair polypeptide is BRCA1, BRCA2, BARD1, PALB2, RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, BLM, RecQ helicase, MRE11, Rad50, NBS1, ATM, ATR, CTIP, Brip, RPA, RPA-like polypeptide, and combinations thereof.
5. The method of claim 3 , wherein long tract gene conversion is detected by detecting an alteration in fluorescence between the cell and the reference cell.
6. The method of claim 5 , wherein the functional significance of a mutation in a DNA repair polypeptide in a biological sample indicates the subject has or has a propensity to develop cancer.