Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro
Open accessAbstract
The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro , which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.
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The PCNA-like 9-1-1 complex (Rad9-Rad1-Hus1) is recruited preferentially to the 5'-primed single-stranded/double-stranded DNA junction by RPA and the Rad17-RFC clamp loader.
“The PCNA-like clamp 9-1-1 complex (Rad9-Rad1-Hus1) is recruited preferentially to the 5′-primed ssDNA/dsDNA junction by RPA and the clamp loader Rad17-RFC complex [102–105].”
From Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress (2014) · cites this paper as [104]
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- Authors
- Bermudez, V. P., Lindsey-Boltz, L. A., Cesare, A. J., Maniwa, Y., Griffith, J. D., Hurwitz, J., & Sancar, A.
- Affiliations
- University of North Carolina at Chapel Hill (US)
- Program in Molecular Biology, Sloan–Kettering Institute, Memorial Sloan–Kettering Cancer Center, New York, NY 10021; and Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2003-02-10
- Volume
- 100
- Issue
- 4
- Pages
- 1633-1638
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC149884
- OpenAlex
- W2034033535
- Cited by
- 324
- References
- 26
- Citations by year
- 2026: 4 · 2025: 5 · 2024: 6 · 2023: 4 · 2022: 8 · 2021: 12 · 2020: 10 · 2019: 7 · 2018: 6 · 2017: 6 · 2016: 12 · 2015: 12 · 2014: 14 · 2013: 14 · 2012: 6
- FWCI
- 10.34
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion)
- UNC Libraries
- Funders
- National Institutes of Health (GM38559)
- National Institute of General Medical Sciences (R01GM032833, GM-31819, R01GM031819, GM32833, F32GM020830, F32-GM20830, GM 38559)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- DNA damage checkpoint, DNA damage sensor, ATP-dependent DNA binding, DNA priming
- MeSH terms
- Adenosine Triphosphate, Cell Cycle, DNA, DNA-Binding Proteins, Humans, Microscopy, Electron, Recombinant Proteins, Cell Cycle Proteins, Replication Protein C
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