Journal Article

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

Diamant, Noam, Geacintov, Nicholas E., Tateishi, Satoshi, Krijger, Peter H. L., Goren, Zohar, Langerak, Petra, Carell, Thomas, Myung, Kyungjae, Kim, Jungmin, D'Andrea, Alan, Jacobs, Heinz, Reißner, Thomas, Lee, Kyoo-young, Hendel, Ayal, Livneh, Zvi

2011

Overview

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

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Animals / Biology / Biomedical research / Brewer's yeast / Cancer / Cisplatin - pharmacology / Cyclin-dependent kinases / Deoxyribonucleic acid / DNA / DNA - biosynthesis / DNA - drug effects / DNA - genetics / DNA Damage / DNA Repair / DNA Replication / DNA synthesis / DNA, Single-Stranded - biosynthesis / DNA, Single-Stranded - genetics / E coli / Experiments / Genetic aspects / Mice / Mutagenesis / Mutation / Physiological aspects / Plasmids / Proliferating Cell Nuclear Antigen - genetics / Proliferating Cell Nuclear Antigen - metabolism / Proteins / Ubiquitin - genetics / Ubiquitin - metabolism / Ubiquitin-proteasome system / Ubiquitination / Ultraviolet Rays