REV7-mediated polyubiquitination and degradation of human REV1

Abstract

REV1 is an evolutionarily conserved Y-family DNA polymerase involved in DNA damage response. Here we demonstrate that human REV1 protein undergoes polyubiquitination and proteosome-dependent degradation. REV1 is an unstable enzyme with high protein turnover. However, REV1 protein can be stabilized by proteasome inhibitors such as MG132 and PS1. Transient transfection of ubiquitin showed that hREV1 is polyubiquitinated. Subsequent to this modification the steady state protein level of REV1 decreased, suggesting that polyubiquitination targets REV1 to proteasome-mediated degradation. REV7, also called MAD2B, is a homolog of mitotic checkpoint protein MAD2 and it interacts directly with REV1. Both REV7 and MAD2 associate with and regulate CDC20, an activator of anaphase promoting complex (APC). Suppression of REV7 expression by shRNA considerably reduced REV1 polyubiquitination, whereas overexpression of CDC20 promoted polyubiquitination of REV1 and its subsequent degradation by proteasome. Upon UV irradiation or treatment with a radiomimetic drug bleomycin, polyubiquitination of REV1 is prohibited. Our findings provide an explanation to a physiologically relevant scenario in which UV irradiation suppresses REV1 polyubiquitination, hence making more REV1 available for translesion synthesis (TLS). On the other hand, given its low fidelity, REV1 is sustained to a minimal level by polyubiquitination to prevent its interference with DNA replication by replicative DNA polymerases. Our work reveals a new mechanism for regulated degradation of a TLS polymerase.