Genetic analysis of DNA-damage tolerance pathways in Arabidopsis

Genetic analysis revealed a two-branch DNA-damage tolerance mechanism in Arabidopsis, namely translesion DNA synthesis and error-free lesion bypass, represented by Rev3 and Rad5a-Uev1C/D, respectively. DNA-damage tolerance (DDT) is a mechanism by which cells complete replication in the presence of replication-blocking lesions. In budding yeast, DDT is achieved through Rad6-Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA), which promotes translesion DNA synthesis (TLS) and is followed by Ubc13-Mms2-Rad5 mediated K63-linked PCNA polyubiquitination that promotes error-free lesion bypass. Arabidopsis and other known plant genomes contain all of the above homologous genes except RAD18, and whether plants possess an intact DDT mechanism is unclear. In this study, we created Arabidopsis UEV1 (homologous to yeast MMS2) gene mutations and obtained two sets of double mutant lines Atuev1ab and Atuev1cd. It turned out that the Atuev1cd, but not the Atuev1ab mutant, was sensitive to DNA damage. Genetic analyses revealed that AtUEV1C/D and AtRAD5a function in the same pathway, while TLS represented by AtREV3 functions in a separate pathway in response to replication-blocking lesions. Furthermore, unlike budding yeast RAD5 that also functions in the TLS pathway, AtRAD5a is not required for TLS. Observations in this study collectively establish a two-branch DDT model in plants with similarity to and difference from the yeast DDT.