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. Copyright (c) 2010 Elsevier Ltd. All rights reserved.