DNA Damage by the Enediyne C-1027 Results in the Inhibition of DNA Replication by Loss of Replication Protein A Function and Activation of DNA-Dependent Protein Kinase†

Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.

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The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: Replication protein A hyperphosphorylation and p53 induction

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.93.24.13825 ◽

1996 ◽

Vol 93 (24) ◽

pp. 13825-13830 ◽

Cited By ~ 60

Author(s):

L. M. Fried ◽

C. Koumenis ◽

S. R. Peterson ◽

S. L. Green ◽

P. van Zijl ◽

...

Keyword(s):

Dna Damage ◽

Protein Kinase ◽

Dna Damage Response ◽

Scid Mouse ◽

Protein A ◽

Replication Protein A ◽

Dependent Protein Kinase ◽

Damage Response ◽

Dependent Protein ◽

Mouse Cells

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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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