Abstract
Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη−/−, POLι−/−, POLκ−/−, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη−/− cells, but not POLι−/− or POLκ−/− cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη−/− cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.
Highlights
Genomic DNA, the genetic blueprint of life, is replicated with remarkably high fidelity by replicative polymerases Polα, Polδ, and Polε to precisely maintain genetic information [1,2,3,4]
triple knockout (TKO) cells were more sensitive to UV, CDDP, and methyl methanesulfonate (MMS) than were the wild-type cells (Fig 1)
We investigated the relative contributions of the Y-family polymerases Polη, Polι, and Polκ in bypass replication of three distinct types of DNA damage
Summary
Genomic DNA, the genetic blueprint of life, is replicated with remarkably high fidelity by replicative polymerases Polα, Polδ, and Polε to precisely maintain genetic information [1,2,3,4]. The DNA replication fork is frequently stalled by damages induced by external factors (such as ultraviolet [UV] light from sunlight and environmental chemicals) and by internal factors (such as oxygen radicals and aldehyde resulting from metabolic reactions). Stalled replication forks are restarted by DNA-damage tolerance (DDT) pathways that bypass DNA. Genetic analysis of Y-family polymerases in human TK6 cells design, data collection and analysis, decision to publish, or preparation of the manuscript
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