Opposite roles of Rad5 in DNA damage tolerance: playing in both error-free and mutagenic lesion bypass

Abstract

DNA Damage Tolerance (DDT) functions to bypass replication-blocking lesions and is divided into two distinct pathways: error-prone Translesion Synthesis (TLS) and error-free Damage Avoidance (DA). Rad5 is a a multifunctional protein that is involved in these DDT processes. Rad5 contains three well defined domains: a RING domain that promotes PCNA polyubiquitination, a ssDNA-dependent ATPase/helicase domain, and a Rev1-binding domain. Both the RING domain and the ATPase/helicase domain are conserved in human Rad5 ortholog HLTF. In this study we used domain-specific mutants to address the contribution of each of Saccharomyces cerevisiae Rad5 functions to lesion tolerance. Using an assay based on the insertion of a single lesion into a defined locus in the genome of a living yeast cell, we demonstrate that Rad5 plays two opposite roles in lesion tolerance: i) Rad5 favors error-free lesion bypass by activating template switching through polyubiquitination of PCNA; ii) Rad5 is required for TLS by recruiting the TLS polymerase Rev1. We also show that the helicase activity does not play any role in lesion tolerance.