Journal article
PCNA Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in Mammalian Cells
PLoS Genetics, Vol.7(9), e1002262
Sep/2011
Abstract
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.
Details
- Title
- PCNA Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in Mammalian Cells
- Creators
- Ayal Hendel (null) - 972WIS_INST___112Peter H. L. Krijger (null)Noam Diamant (null) - 972WIS_INST___112Zohar Goren (null) - 972WIS_INST___112Petra Langerak (null)Jungmin Kim (null)Thomas Reissner (null)Kyoo-young Lee (null)Nicholas E. Geacintov (null)Thomas Carell (null)Kyungjae Myung (null)Satoshi Tateishi (null)Alan D'Andrea (null)Heinz Jacobs (null)Zvi Livneh (null) - 972WIS_INST___112
- Resource Type
- Journal article
- Publication Details
- PLoS Genetics, Vol.7(9), e1002262; Sep/2011
- Number of pages
- 13
- Language
- English
- DOI
- https://doi.org/10.1371/journal.pgen.1002262
- Grant note
- Flight Attendant Medical Research Institute; Leona M. and Harry B. Helmsley Charitable Trust; Israel Science Foundation [1136/08]; Netherlands Organization for Scientific Research; Dutch Cancer Foundation [917.56.328, 2008-4112]; NIH USA [CA099194]ZL is the incumbent of the Maxwell Ellis Professorial Chair in Biomedical Research. This work was supported by grants to ZL from the Flight Attendant Medical Research Institute, the Leona M. and Harry B. Helmsley Charitable Trust, and the Israel Science Foundation (no. 1136/08); to HJ from The Netherlands Organization for Scientific Research and the Dutch Cancer Foundation (VIDI program 917.56.328 and KWF 2008-4112); and to NEG from NIH USA (no. CA099194). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript._ALMAME_DELIMITER_
- Record Identifier
- 993265944403596
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