DNA replication-coupled PCNA mono-ubiquitination and polymerase switching in a human in vitro system.
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 delta on PCNA mono-ubiquitination with the ubiquitin-conjugating enzyme and ligase UBE2A/HHR6A/RAD6A-RAD18. The results revealed that PCNA interacting with pol delta 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 delta and a translesion pol, pol eta, 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 eta. This study provides a framework for detailed analyses of molecular mechanisms of human pol switching and regulation of translesion DNA synthesis.
Masuda
Yuji
Y
Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. masudayu@hiroshima-u.ac.jp
Piao
Jinlian
J
Kamiya
Kenji
K
D004247:DNA / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D006801:Humans; D008954:Models, Biological; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D044767:Ubiquitin-Protein Ligases; D004258:DNA Polymerase III