Abstract
DNA repair mechanisms maintain genomic integrity upon exposure to various types of DNA damage, which cause either single- or double-strand breaks in the DNA. Here, we propose a strategy for the functional study of single nucleotide polymorphisms (SNPs) in the human DNA repair genes XPD/ERCC2, RAD18, and KU70/XRCC6 and the checkpoint activation gene ATR that are essentially involved in the cell cycle and DNA damage repair. We analyzed the mutational effects of the DNA repair genes under DNA-damaging conditions, including ultraviolet irradiation and treatment with genotoxic reagents, using a Saccharomyces cerevisiae system to overcome the limitations of the human cell-based assay. We identified causal variants from selected SNPs in the present analyses. (i) R594C SNP in RAD3 (human XPD/ERCC2) caused severe reductions in the growth rate of mutant cells upon short-wavelength UV irradiation or chemical reagent treatment. (ii) The growth rates of the selected variants in RAD18, YKU70, and MEC1 were similar to those of wild-type cells on methyl methanesulfonate and hydroxyurea treated media. (iii) We also assessed the structural impact of the SNPs by analyzing differences in the structural conformation and calculating the root mean square deviation, which is a measure of the discordance of the Cα atoms between protein structures. Based on the above results, we propose that these analytical approaches serve as efficient methods for the identification of causal variants of human disease-causing genes and elucidation of yeast-cell based molecular mechanisms.
Highlights
The maintenance of genomic integrity, mediated by DNA repair pathways, is critical for proper cell cycle progression and cell proliferation
To identify variants in the human DNA repair genes that can potentially cause diseases such as cancer, we compared the homologous regions of various DNA repair genes between humans and yeast
Candidate single nucleotide polymorphisms (SNPs) were selected based on data obtained from dbSNPs
Summary
The maintenance of genomic integrity, mediated by DNA repair pathways, is critical for proper cell cycle progression and cell proliferation. Nine SNPs in four different DNA repair genes, ATR, ERCC2, RAD18, and XRCC6, were selected for further analysis (Table 1, Fig 1). To examine the growth characteristics of yeast cells harboring mutations in the MEC1 gene, mutant strains were cultured on yeast extract-peptone-dextrose (YPD) plates, treated with various DNA damaging agents, and subsequently subjected to growth assays (Fig 2A).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.