scholarly journals The Mre11 Complex Mediates the S-Phase Checkpoint through an Interaction with Replication Protein A

2007 ◽  
Vol 27 (17) ◽  
pp. 6053-6067 ◽  
Author(s):  
Erin Olson ◽  
Christian J. Nievera ◽  
Enbo Liu ◽  
Alan Yueh-Luen Lee ◽  
Longchuan Chen ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Protein A ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Initiation ◽  
Replication Protein ◽  
Direct Role ◽  
Origin Firing ◽  
S Phase Checkpoint

ABSTRACT The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.

Phosphorylation of replication protein A by S-phase checkpoint kinases

DNA Repair ◽  
2006 ◽  
Vol 5 (3) ◽  
pp. 369-380 ◽  
Author(s):  
Jen-Sing Liu ◽  
Shu-Ru Kuo ◽  
Thomas Melendy
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein ◽  
Checkpoint Kinases ◽  
S Phase Checkpoint

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

2000 ◽  
Vol 20 (8) ◽  
pp. 2696-2705 ◽  
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.

scholarly journals Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

2008 ◽  
Vol 19 (10) ◽  
pp. 4374-4382 ◽  
Author(s):  
Ling Yin ◽  
Alexandra Monica Locovei ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Damage Checkpoint ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Fork Stalling ◽  
S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

scholarly journals Activation of mammalian Chk1 during DNA replication arrest

2001 ◽  
Vol 154 (5) ◽  
pp. 913-924 ◽  
Author(s):  
Carmen Feijoo ◽  
Clare Hall-Jackson ◽  
Rong Wu ◽  
David Jenkins ◽  
Jane Leitch ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
S Phase ◽  
Global Pattern ◽  
Origin Firing ◽  
Replication Arrest ◽  
Molecule Inhibitor ◽  
Replication Block ◽  
Phase Progression ◽  
S Phase Checkpoint

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

scholarly journals A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

2008 ◽  
Vol 284 (9) ◽  
pp. 5807-5818 ◽  
Author(s):  
Darren E. Casteel ◽  
Shunhui Zhuang ◽  
Ying Zeng ◽  
Fred W. Perrino ◽  
Gerry R. Boss ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Mammalian Cells ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein

scholarly journals SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication

2020 ◽  
Vol 48 (7) ◽  
pp. 3657-3677 ◽  
Author(s):  
Nichodemus O Onwubiko ◽  
Angela Borst ◽  
Suraya A Diaz ◽  
Katharina Passkowski ◽  
Felicia Scheffel ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Dissociation Constants ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Replication Initiation ◽  
Replication Protein ◽  
Central Process ◽  
Lagging Strand

Abstract DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10–30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

scholarly journals Stabilization of a G-Quadruplex from Unfolding by Replication Protein A Using Potassium and the Porphyrin TMPyP4

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Aishwarya Prakash ◽  
Fabien Kieken ◽  
Luis A. Marky ◽  
Gloria E. O. Borgstahl
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Thermal Stabilization ◽  
Replication Protein A ◽  
Replication Protein ◽  
Unique Problem ◽  
Ssdna Binding ◽  
Binding Domains ◽  
G Quadruplex ◽  
Stabilization Effect

Replication protein A (RPA) plays an essential role in DNA replication by binding and unfolding non-canonical single-stranded DNA (ssDNA) structures. Of the six RPA ssDNA binding domains (labeled A-F), RPA-CDE selectively binds a G-quadruplex forming sequence (5′-TAGGGGAAGGGTTGGAGTGGGTT-3′called Gq23). In K+, Gq23 forms a mixed parallel/antiparallel conformation, and in Na+Gq23 has a less stable (TMlowered by ∼20∘C), antiparallel conformation. Gq23 is intramolecular and 1D NMR confirms a stable G-quadruplex structure in K+. Full-length RPA and RPA-CDE-core can bind and unfold the Na+form of Gq23 very efficiently, but complete unfolding is not observed with the K+form. Studies with G-quadruplex ligands, indicate that TMPyP4 has a thermal stabilization effect on Gq23 in K+, and inhibits complete unfolding by RPA and RPA-CDE-core. Overall these data indicate that G-quadruplexes present a unique problem for RPA to unfold and ligands, such as TMPyP4, could possibly hinder DNA replication by blocking unfolding by RPA.

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