Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae.
Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer.
Department of Life Science, Chung-Ang University, Seoul, Korea.
Min-Soo
MS
Lee
Department of Life Science, Chung-Ang University, Seoul, Korea.
Mi
M
Yu
Department of Biomedical Science, CHA University, Seongnam, Korea.
Kyoung-Yeon
KY
Kim
Department of Life Science, Chung-Ang University, Seoul, Korea.
Geun-Hee
GH
Park
Department of Biomedical Science, CHA University, Seongnam, Korea.
KyuBum
K
Kwack
Department of Life Science, Chung-Ang University, Seoul, Korea.
Keun P
KP
Kim
D000483:Alleles; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D014644:Genetic Variation; D059765:Homologous Recombination / Q000235:genetics*; D006801:Humans; D050296:Microbial Viability; D009154:Mutation / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013172:Spores, Fungal / Q000502:physiology
D029701:Saccharomyces cerevisiae Proteins