Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract

The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI,discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.

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Effect of Topoisomerase Inhibitors on the Formation of Replication Protein A-Foci in Nuclei Reconstituted in Xenopus Egg Extract

JOURNAL OF HEALTH SCIENCE ◽

10.1248/jhs.48.296 ◽

2002 ◽

Vol 48 (3) ◽

pp. 296-301

Author(s):

Takayuki Kobayashi ◽

Shusuke Tada ◽

Takashi Tsuyama ◽

Hiromu Murofushi ◽

Masayuki Seki ◽

...

Keyword(s):

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Topoisomerase Inhibitors ◽

Xenopus Egg Extract ◽

Egg Extract ◽

Xenopus Egg

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The Cellular Response to DNA Damage Induced by the Enediynes C-1027 and Neocarzinostatin Includes Hyperphosphorylation and Increased Nuclear Retention of Replication Protein A (RPA) and Trans Inhibition of DNA Replication†

Biochemistry ◽

10.1021/bi001668t ◽

2001 ◽

Vol 40 (15) ◽

pp. 4792-4799 ◽

Cited By ~ 8

Author(s):

Mary M. McHugh ◽

Xia Yin ◽

Shu-Ru Kuo ◽

Jen-Sing Liu ◽

Thomas Melendy ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Cellular Response ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Nuclear Retention

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DNA Replication but Not Nucleotide Excision Repair Is Required for UVC-Induced Replication Protein A Phosphorylation in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2696-2705.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2696-2705 ◽

Cited By ~ 23

Author(s):

Gregory Rodrigo ◽

Sophie Roumagnac ◽

Marc S. Wold ◽

Bernard Salles ◽

Patrick Calsou

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Nucleotide Excision Repair ◽

Mammalian Cells ◽

Excision Repair ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

Dependent Manner ◽

Nucleotide Excision

ABSTRACT Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.

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Functions of human replication protein A (RPA): From DNA replication to DNA damage and stress responses

Journal of Cellular Physiology ◽

10.1002/jcp.20622 ◽

2006 ◽

Vol 208 (2) ◽

pp. 267-273 ◽

Cited By ~ 192

Author(s):

Yue Zou ◽

Yiyong Liu ◽

Xiaoming Wu ◽

Steven M. Shell

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Stress Responses ◽

Protein A ◽

Replication Protein A ◽

Replication Protein

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Human RPA activates BLM’s bidirectional DNA unwinding from a nick

eLife ◽

10.7554/elife.54098 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Zhenheng Qin ◽

Lulu Bi ◽

Xi-Miao Hou ◽

Siqi Zhang ◽

Xia Zhang ◽

...

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Single Molecule ◽

Genome Stability ◽

Protein A ◽

Replication Protein A ◽

Replication Protein ◽

High Abundance ◽

Dna Unwinding ◽

Dna Replication And Repair

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

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