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Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae.

Lee MS, Yu M, Kim KY, Park GH, Kwack K, Kim KP - PLoS ONE (2015)

Bottom Line: Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively.Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes.The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Chung-Ang University, Seoul, Korea.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Schematic diagram of experiments and the selection of SNPs in human RAD51 and RAD52 genes.(A) Diagram indicating the method of SNP selection. The SNPs in hRAD51 and hRAD52 were selected using the National Center for Biotechnology Information Single Nucleotide Polymorphism Database (NCBI dbSNP), (http://www.ncbi.nlm.nih.gov/snp/). (B) Missense SNPs located in functional domains were compared with genetic variations using SIFT and PolyPhen. “Deleterious” and “Probably damaging” allelic mutants might affect function via structural alterations of Rad51 and Rad52. (C) Comparison of the functional domains of Rad51 in humans and yeast [30]. * regions containing rad51-F317I and rad51-K371Q mutations. (D) Comparison of the functional domains of Rad52 in humans and yeast [31, 32]. * regions containing rad52-G41R, rad52-R52W, and rad52-G107C mutations.
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pone.0124152.g001: Schematic diagram of experiments and the selection of SNPs in human RAD51 and RAD52 genes.(A) Diagram indicating the method of SNP selection. The SNPs in hRAD51 and hRAD52 were selected using the National Center for Biotechnology Information Single Nucleotide Polymorphism Database (NCBI dbSNP), (http://www.ncbi.nlm.nih.gov/snp/). (B) Missense SNPs located in functional domains were compared with genetic variations using SIFT and PolyPhen. “Deleterious” and “Probably damaging” allelic mutants might affect function via structural alterations of Rad51 and Rad52. (C) Comparison of the functional domains of Rad51 in humans and yeast [30]. * regions containing rad51-F317I and rad51-K371Q mutations. (D) Comparison of the functional domains of Rad52 in humans and yeast [31, 32]. * regions containing rad52-G41R, rad52-R52W, and rad52-G107C mutations.

Mentions: We selected 12 single nucleotide polymorphisms (SNPs) within RAD51 (8 SNPs) and RAD52 (4 SNPs) from the NCBI SNP database (S2 Table). We compared the homologs of these two genes in humans and yeast, but excluded several SNPs that were synonymous. The PolyPhen (http://genetics.bwh.harvard.edu/pph) and SIFT (http://sift.jcvi.org) programs were used to predict the effects of amino acid substitutions on protein function and structure, respectively (Fig 1A). From these analyses, we predicted that the RAD51 SNPs, rad51-F259I and rad51-K313Q, and the RAD52 SNPs, rad52-G59R, rad52-R70W, and rad52-G125C lead to the formation of non-functional proteins that would likely be non-functional in physiological conditions (Fig 1B). Specifically, these missense SNPs were located in the ATPase domains of Rad51 and DNA-binding domains of Rad52 that affect HR activity; these functional domains are highly conserved between humans and yeast (Fig 1C and 1D).


Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae.

Lee MS, Yu M, Kim KY, Park GH, Kwack K, Kim KP - PLoS ONE (2015)

Schematic diagram of experiments and the selection of SNPs in human RAD51 and RAD52 genes.(A) Diagram indicating the method of SNP selection. The SNPs in hRAD51 and hRAD52 were selected using the National Center for Biotechnology Information Single Nucleotide Polymorphism Database (NCBI dbSNP), (http://www.ncbi.nlm.nih.gov/snp/). (B) Missense SNPs located in functional domains were compared with genetic variations using SIFT and PolyPhen. “Deleterious” and “Probably damaging” allelic mutants might affect function via structural alterations of Rad51 and Rad52. (C) Comparison of the functional domains of Rad51 in humans and yeast [30]. * regions containing rad51-F317I and rad51-K371Q mutations. (D) Comparison of the functional domains of Rad52 in humans and yeast [31, 32]. * regions containing rad52-G41R, rad52-R52W, and rad52-G107C mutations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418691&req=5

pone.0124152.g001: Schematic diagram of experiments and the selection of SNPs in human RAD51 and RAD52 genes.(A) Diagram indicating the method of SNP selection. The SNPs in hRAD51 and hRAD52 were selected using the National Center for Biotechnology Information Single Nucleotide Polymorphism Database (NCBI dbSNP), (http://www.ncbi.nlm.nih.gov/snp/). (B) Missense SNPs located in functional domains were compared with genetic variations using SIFT and PolyPhen. “Deleterious” and “Probably damaging” allelic mutants might affect function via structural alterations of Rad51 and Rad52. (C) Comparison of the functional domains of Rad51 in humans and yeast [30]. * regions containing rad51-F317I and rad51-K371Q mutations. (D) Comparison of the functional domains of Rad52 in humans and yeast [31, 32]. * regions containing rad52-G41R, rad52-R52W, and rad52-G107C mutations.
Mentions: We selected 12 single nucleotide polymorphisms (SNPs) within RAD51 (8 SNPs) and RAD52 (4 SNPs) from the NCBI SNP database (S2 Table). We compared the homologs of these two genes in humans and yeast, but excluded several SNPs that were synonymous. The PolyPhen (http://genetics.bwh.harvard.edu/pph) and SIFT (http://sift.jcvi.org) programs were used to predict the effects of amino acid substitutions on protein function and structure, respectively (Fig 1A). From these analyses, we predicted that the RAD51 SNPs, rad51-F259I and rad51-K313Q, and the RAD52 SNPs, rad52-G59R, rad52-R70W, and rad52-G125C lead to the formation of non-functional proteins that would likely be non-functional in physiological conditions (Fig 1B). Specifically, these missense SNPs were located in the ATPase domains of Rad51 and DNA-binding domains of Rad52 that affect HR activity; these functional domains are highly conserved between humans and yeast (Fig 1C and 1D).

Bottom Line: Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively.Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes.The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Chung-Ang University, Seoul, Korea.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus