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

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 re...

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Published in:PLoS genetics Vol. 7; no. 9; p. e1002262
Main Authors: Hendel, Ayal, Krijger, Peter H. L., Diamant, Noam, Goren, Zohar, Langerak, Petra, Kim, Jungmin, Reißner, Thomas, Lee, Kyoo-young, Geacintov, Nicholas E., Carell, Thomas, Myung, Kyungjae, Tateishi, Satoshi, D'Andrea, Alan, Jacobs, Heinz, Livneh, Zvi
Format: Journal Article
Language:English
Published: United States Public Library of Science 01.09.2011
Public Library of Science (PLoS)
Subjects:
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
Japan
ISSN:1553-7404, 1553-7390, 1553-7404
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Summary: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.
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Conceived and designed the experiments: AH PHLK ND ZG. Performed the experiments: AH PHLK ND ZG. Analyzed the data: AH PHLK ND ZG HJ ZL. Contributed reagents/materials/analysis tools: PL JK TR KL NEG TC KM ST AD HJ. Wrote the paper: ZL AH.
These authors also contributed equally to this work.
ISSN:1553-7404
1553-7390
1553-7404
DOI:10.1371/journal.pgen.1002262