| Research Abstract |
Damaged DNA can be a serious threat to successful completion of DNA replication. If a replication fork encounters a damage on the template DNA it arrest, resulting in a large gap on daughter strand and occasionally a break. These lesions are processed by a mechanism called postreplication repair (PRR) that enables the DNA replication without removing the lesions. Two different repair pathways are thought to mediate PRR. Homologous recombination(HR) uses an undamaged sister duplex as a template to repair gaps and breaks. Translesion DNA synthesis (TLS), controlled by a putative E3 ubiquitine ligase Rad18 and E2 enzyme Rad6,relies on non-essential DNA polymerases such as polζ (Rev3/Rev7) and polη (Rad30) to synthesize DNA across the lesion. To study the molecular basis of PRR in vertebrates, we have established single and multiple knockout-mutants of Rev1,Rev3,Rev7,Rad18,Rad30,PolK,PolQ(involved in TLS) and Rad51 paralog and Rad54 (involved in HR) genes using chicken DT40 cells.
HR defect
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ive mutants showed a decreased level of sister chromatid exchange (SCE) which reflects a DNA repair process by crossover-type HR and accumulated spontaneous chromosomal breaks. In contrast, TLS mutants including rev1,rev3,rev7, polκ and rad18 mutants showed a significant increase in SCE, suggesting that in the absence of TLS,HR predominates to bypass the spontaneous damages. rev and rad18 mutants were highly sensitive to a wide range of DNA-damaging agents, including UV, ionizing radiation, MMS and cross-linking agents. rev single mutants and rev1/3/7 triple mutant showed almost comparable sensitivities against various genotoxic stresses, suggesting that Rev1 and polζ (Rev3/7) function as a functional unit in DNA damage response. On the other hand, rad30 and polκ showed the sensitivity only to UV, suggesting that each TLS polymerase play a distinctive role in DNA damage response. Interestingly, rev1 and rad30 but not rev3 mutant exhibited a significant decrease in the frequency of immunoglobulin gene conversion, suggesting the participation of these molecules in a recombination-based pathway, such as DNA synthesis step of intragenic homologous recombination. Thus, the molecules involved in TLS pathway play a variety of roles in DNA repair and recombination. Less
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