PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells

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

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere1002262
JournalPLoS Genetics
Volume7
Issue number9
DOIs
StatePublished - Sep 2011

Fingerprint

Ubiquitination
Proliferating Cell Nuclear Antigen
DNA
synthesis
cells
lesions (animal)
lesion
cisplatin
DNA-Directed DNA Polymerase
ubiquitination
Cisplatin
Ubiquitin-Protein Ligases
ubiquitin-protein ligase
single-stranded DNA
Single-Stranded DNA
Ubiquitin
DNA-directed DNA polymerase
ubiquitin
Mutagenesis
DNA Damage

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Ecology, Evolution, Behavior and Systematics
  • Cancer Research
  • Genetics(clinical)

Cite this

Hendel, A., Krijger, P. H. L., Diamant, N., Goren, Z., Langerak, P., Kim, J., ... Livneh, Z. (2011). PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells. PLoS Genetics, 7(9), [e1002262]. https://doi.org/10.1371/journal.pgen.1002262

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells. / Hendel, Ayal; Krijger, Peter H L; Diamant, Noam; Goren, Zohar; Langerak, Petra; Kim, Jungmin; Reißner, Thomas; Lee, Kyoo young; Geacintov, Nicholas; Carell, Thomas; Myung, Kyungjae; Tateishi, Satoshi; D'Andrea, Alan; Jacobs, Heinz; Livneh, Zvi.

In: PLoS Genetics, Vol. 7, No. 9, e1002262, 09.2011.

Research output: Contribution to journalArticle

Hendel, A, Krijger, PHL, Diamant, N, Goren, Z, Langerak, P, Kim, J, Reißner, T, Lee, KY, Geacintov, N, Carell, T, Myung, K, Tateishi, S, D'Andrea, A, Jacobs, H & Livneh, Z 2011, 'PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells', PLoS Genetics, vol. 7, no. 9, e1002262. https://doi.org/10.1371/journal.pgen.1002262
Hendel, Ayal ; Krijger, Peter H L ; Diamant, Noam ; Goren, Zohar ; Langerak, Petra ; Kim, Jungmin ; Reißner, Thomas ; Lee, Kyoo young ; Geacintov, Nicholas ; Carell, Thomas ; Myung, Kyungjae ; Tateishi, Satoshi ; D'Andrea, Alan ; Jacobs, Heinz ; Livneh, Zvi. / PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells. In: PLoS Genetics. 2011 ; Vol. 7, No. 9.
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