DNA Replication-Coupled PCNA Mono-Ubiquitination and Polymerase Switching in a Human InVitro System |
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Authors: | Yuji Masuda |
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Institution: | Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan |
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Abstract: | Translesion DNA synthesis is a mechanism of DNA damage tolerance, and mono-ubiquitination of proliferating cell nuclear antigen (PCNA) is considered to play a key role in regulating the switch from replicative to translesion DNA polymerases (pols). In this study, we analyzed effects of a replicative pol δ on PCNA mono-ubiquitination with the ubiquitin-conjugating enzyme and ligase UBE2A/HHR6A/RAD6A-RAD18. The results revealed that PCNA interacting with pol δ is a better target for ubiquitination, and PCNA mono-ubiquitination could be coupled with DNA replication. Consequently, we could reconstitute replication-coupled switching between pol δ and a translesion pol, pol η, on an ultraviolet-light-irradiated template. With this system, we obtained direct evidence that polymerase switching reactions are stimulated by mono-ubiquitination of PCNA, depending on a function of the ubiquitin binding zinc finger domain of pol η. This study provides a framework for detailed analyses of molecular mechanisms of human pol switching and regulation of translesion DNA synthesis. |
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Keywords: | E1 ubiquitin-activating enzyme E1 PCNA proliferating cell nuclear antigen pol DNA polymerase PRR post-replication repair RFC replication factor C RF replication factor RPA replication protein A ssDNA single-stranded DNA TLS translesion DNA synthesis TS template switching UE ubiquitin enzyme UBZ ubiquitin binding zinc finger BSA bovine serum albumin EDTA ethylenediaminetetraacetic acid |
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