Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress
Open Access
- 15 November 2009
- journal article
- Published by The Company of Biologists in Journal of Cell Science
- Vol. 122 (22), 4070-4080
- https://doi.org/10.1242/jcs.053702
Abstract
ATR is an essential kinase activated in response to DNA-replication stress, with a known target being the RPA2 subunit of human replication protein A (RPA). We find that S33-RPA2 phosphorylation by ATR occurs primarily in the late-S and G2 phases, probably at sites of residual stalled DNA-replication forks, with S33-P-RPA2 contained within nuclear repair centers. Although cells in which endogenous RPA2 was `replaced' with an RPA2 protein with mutations T21A and S33A (T21A/S33A-RPA) had normal levels of DNA replication under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress. These cells also had abnormally high levels of chromatin-bound RPA, indicative of increased amounts of single-stranded DNA (ssDNA) and showed defective recovery from stress. Cells replaced with the mutant RPA2 also generated G1 cells with a broader DNA distribution and high levels of apoptosis following stress, compared with cells expressing wild-type RPA2. Surprisingly, cells expressing the wild-type RPA2 subunit had increased levels of stress-dependent DNA breaks. Our data demonstrate that RPA phosphorylation at the T21 and S33 sites facilitates adaptation of a DNA-replication fork to replication stress.Keywords
This publication has 48 references indexed in Scilit:
- RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damageProceedings of the National Academy of Sciences of the United States of America, 2008
- Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA RepairPublished by Elsevier BV ,2007
- NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapseJournal of Cell Science, 2007
- The Intra-S-Phase Checkpoint Affects both DNA Replication Initiation and Elongation: Single-Cell and -DNA Fiber AnalysesMolecular and Cellular Biology, 2007
- The DNA damage response during an unperturbed S-phaseDNA Repair, 2007
- The Human Tim/Tipin Complex Coordinates an Intra-S Checkpoint Response to UV That Slows Replication Fork DisplacementMolecular and Cellular Biology, 2007
- Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication CentersMolecular and Cellular Biology, 2004
- Bloom syndrome cells undergo p53-dependent apoptosis and delayed assembly of BRCA1 and NBS1 repair complexes at stalled replication forksThe Journal of cell biology, 2003
- RPA Phosphorylation in Mitosis Alters DNA Binding and Protein−Protein InteractionsBiochemistry, 2003
- Stability and Nuclear Distribution of Mammalian Replication Protein A Heterotrimeric ComplexExperimental Cell Research, 2000