Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage
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RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.
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A high-salt wash disrupts protein-protein and protein-DNA interactions, removing XPA from the ATR complex.
“After immunoprecipitation with anti-ATR antibodies, the immunoprecipitates were washed with buffer of high salt concentration (0.6 mol/L NaCl) to disrupt protein-protein and protein-DNA interactions (Fig. 4C, lane 2, shows the loss of XPA from the complex following the high salt wash).”
From Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related–Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation (2006) · cites this paper as [13]
The Figure 5F caption (indexed under Discussion; page unavailable) describes a 600 mM NaCl wash used to remove proteins bound to Rad51 or Rad52 immunoprecipitates. It does not show a high-salt wash of an ATR complex or loss of XPA; the Figure 4 caption instead describes ATR immunoprecipitation followed by detection of RPA32. (LLM, conf=0.85)
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- Authors
- Wu, X., Yang, Z., Liu, Y., & Zou, Y.
- Affiliations
- East Tennessee State University (US)
- Venue
- Biochemical Journal
- Publisher
- Portland Press Ltd.
- Published
- 2005-10-25
- Volume
- 391
- Issue
- 3
- Pages
- 473-480
- ISSN
- 0264-6021 (ISSN-L)
- 1470-8728
- PMC
- PMC1276948
- OpenAlex
- W2063532739
- Cited by
- 81
- References
- 43
- Citations by year
- 2025: 3 · 2024: 4 · 2023: 1 · 2022: 1 · 2021: 5 · 2020: 2 · 2019: 2 · 2017: 3 · 2016: 3 · 2015: 5 · 2014: 5 · 2013: 1 · 2012: 6
- FWCI
- 2.26
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Biochemical Journal (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Digital Commons - East Tennessee State University (East Tennessee State University) (submittedVersion)
- Funders
- East Tennessee State University
- National Cancer Institute (R56CA086927, R01CA086927, CA-86927)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Effects of Radiation Exposure
- Keywords
- replication protein A, DNA double-strand break repair, DNA damage checkpoint, ATR kinase, RAD51, RAD52, protein-protein interaction, UV-induced DNA damage, camptothecin, nuclear foci
- MeSH terms
- Adenocarcinoma, DNA Damage, DNA Repair, Gene Expression Regulation, Humans, Lung Neoplasms, Phosphorylation, Protein Binding, Ultraviolet Rays, Protein Transport, Cell Line, Tumor, Rad51 Recombinase, Replication Protein A, Rad52 DNA Repair and Recombination Protein
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