Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities
Open accessAbstract
Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono- and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.
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UVC-induced γH2AX in Polη-deficient cells is associated with ssDNA generated by uncoupling of replicative DNA polymerase and helicase at stalled forks.
“UVC-induced ␥H2AX in Pol-deficient cells has also been previously associated with the generation of single-stranded DNA (ssDNA) caused by the uncoupling between the replicative DNA polymerase and the helicase at stalled replication forks [23,40,48]”
From Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome (2014) · cites this paper as [48]
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- Authors
- Chang, D. J., Lupardus, P. J., & Cimprich, K. A.
- Affiliations
- Stanford Medicine (US)
- Corresponding author
- Cimprich, Karlene A.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2006-10
- Volume
- 281
- Issue
- 43
- Pages
- 32081-32088
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2020754261
- Cited by
- 101
- References
- 51
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 1 · 2023: 2 · 2022: 2 · 2021: 3 · 2020: 1 · 2019: 4 · 2018: 6 · 2017: 4 · 2016: 5 · 2015: 6 · 2014: 6 · 2013: 7 · 2012: 8
- FWCI
- 3.81
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- California Breast Cancer Research Program
- National Institutes of Health
- National Institute of General Medical Sciences (GM62193, R01GM062193)
- National Institute of Environmental Health Sciences (R01ES016486)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- proliferating cell nuclear antigen, PCNA monoubiquitination, DNA replication stalling, MCM helicase, DNA polymerase, single-stranded DNA, ATRIP, RAD1, DNA damage tolerance, ultraviolet radiation, aphidicolin, Xenopus egg extracts
- MeSH terms
- Animals, Chromosomes, DNA-Directed DNA Polymerase, DNA Replication, DNA Helicases, Enzyme Inhibitors, Female, Models, Biological, Oocytes, Ultraviolet Rays, Xenopus laevis, Aphidicolin, Proliferating Cell Nuclear Antigen, Ubiquitin
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