PCNA Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in Mammalian Cells

• DOI: 10.1371/journal.pgen.1002262
• Publication Date: 2011-09-09T00:34:08Z
• Authors: Ayal Hendel; Peter H. L. Krijger; Noam Diamant; Zohar Goren; Petra Langerak; Jungmin Kim; Thomas Reißner; Kyoo-young Lee; Nicholas E. Geaci...; Thomas Carell; Kyungjae Myung; Satoshi Tateishi; Alan D'Andrea; Heinz Jacobs; Zvi Livneh
• Keywords: DNA polymerase delta; DNA clamp

Translesion DNA synthesis (TLS) is a damage tolerance mechanism in which specialized low-fidelity polymerases bypass replication-blocking lesions, and it usually associated with mutagenesis. In Saccharomyces cerevisiae key event TLS the monoubiquitination of PCNA, enables recruitment to damaged site through their ubiquitin-binding domain. mammals, however, there debate on requirement for ubiquitinated PCNA (PCNA-Ub) TLS. We show that UV-induced Rpa foci, indicative single-stranded (ssDNA) regions caused by UV, accumulate faster disappear more slowly PcnaK164R/K164R cells, are resistant ubiquitination, compared Pcna+/+ consistent defect. Direct analysis these using gapped plasmids site-specific showed strongly reduced across UV lesions cisplatin-induced intrastrand GG crosslink. A similar effect was obtained cells lacking Rad18, E3 ubiquitin ligase monoubiquitinates PCNA. Consistently, Usp1, enzyme de-ubiquitinates exhibited increased lesion cisplatin adduct. contrast, Rad5-homologs Shprh Hltf, polyubiquitinate normal Knocking down expression genes Rev3L, PolH, or Rev1 mouse embryo fibroblasts each an sensitivity radiation, indicating existence pathways independent PCNA-Ub. Taken together results indicate PCNA-Ub required maximal However, can be recruited also absence PCNA-Ub, perform TLS, albeit at significantly lower efficiency altered mutagenic specificity.