Online citations, reference lists, and bibliographies.
← Back to Search

Continued DNA Synthesis In Replication Checkpoint Mutants Leads To Fork Collapse

S. Sabatinos, Marc D. Green, S. Forsburg
Published 2012 · Biology, Medicine

Cite This
Download PDF
Analyze on Scholarcy
ABSTRACT Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork “collapse point” in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.
This paper references
Fork Reversal and ssDNA Accumulation at Stalled Replication Forks Owing to Checkpoint Defects
J. Sogo (2002)
Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication.
M. Pacek (2006)
Mrc 1 protects uncapped budding yeast telomeres from exonuclease EXO 1
DR Williams (2005)
Basis for the Checkpoint Signal Specificity That Regulates Chk1 and Cds1 Protein Kinases
J. Brondello (1999)
Damage Tolerance Protein Mus81 Associates with the FHA1 Domain of Checkpoint Kinase Cds1
M. Boddy (2000)
Genome‐wide characterization of fission yeast DNA replication origins
Christian Heichinger (2006)
ImageJ. National Institutes of Health, Bethesda, MD.
W. S. Rasband (2011)
Mus 81 , Rhp 51 ( Rad 51 ) , and Rqh 1 form an epistatic pathway required for the S - phase DNA damage checkpoint
PY Wu (2009)
Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases.
Giacomo De Piccoli (2012)
DNA replication origins fire stochastically in fission yeast.
P. K. Patel (2006)
Histone H2A
T. M. Nakamura (2004)
Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes
L. Zou (2003)
Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment
S. Janes (2012)
Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1*
T. Tanaka (2010)
Mrc1 Marks Early-Firing Origins and Coordinates Timing and Efficiency of Initiation in Fission Yeast †
M. Hayano (2011)
Checkpoint-mediated control of replisome–fork association and signalling in response to replication pausing
C. Lucca (2004)
Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1
K. Tanaka (2001)
Schizosaccharomyces pombe Cds1Chk2 regulates homologous recombination at stalled replication forks through the phosphorylation of recombination protein Rad60
Izumi Miyabe (2009)
Mus81, Rhp51(Rad51), and Rqh1 form an epistatic pathway required for the S-phase DNA damage checkpoint.
Nicholas A. Willis (2009)
Rhp51-Dependent Recombination Intermediates That Do Not Generate Checkpoint Signal Are Accumulated in Schizosaccharomyces pombe rad60 and smc5/6 Mutants after Release from Replication Arrest
Izumi Miyabe (2006)
Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication
W. Feng (2006)
Mcm4 C-terminal domain of MCM helicase prevents excessive formation Sabatinos et al. 4996 Molecular and Cellular Biology of single-stranded DNA at stalled replication forks
N Nitani (2008)
Regulation of ribonucleotide reductase.
A. Holmgren (1981)
S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex
Y. Katou (2003)
Schizosaccharomyces pombe Hsk1p Is a Potential Cds1p Target Required for Genome Integrity
H. A. Snaith (2000)
Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S‐phase checkpoint
C. Alabert (2009)
Mrc1 transduces
K. Furuya (2001)
Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.
T. S. Byun (2005)
Mcl1p Is a Polymerase α Replication Accessory Factor Important for S-Phase DNA Damage Survival
D. R. Williams (2005)
Mrc1 marks early
M. Hayano (2011)
Replication Stress-Induced Chromosome Breakage Is Correlated with Replication Fork Progression and Is Preceded by Single-Stranded DNA Formation
W. Feng (2011)
A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication
C. Santocanale (1998)
Minichromosome Maintenance Proteins Interact with Checkpoint and Recombination Proteins To Promote S-Phase Genome Stability
J. Bailis (2008)
Hydroxyurea Arrests DNA Replication by a Mechanism That Preserves Basal dNTP Pools*
Ahmet Koç (2004)
Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway
D. Nakada (2004)
Genome-wide localization of small molecules.
Lars Anders (2014)
Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover
Thomas Robert (2006)
ATRIP associates with replication protein A-coated ssDNA through multiple interactions.
Yuka Namiki (2006)
Temporal separation of replication and recombination requires the intra-S checkpoint
P. Meister (2005)
Genomic mapping
Fangman (2006)
Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks
Naoki Nitani (2008)
Swi1 Prevents Replication Fork Collapse and Controls Checkpoint Kinase Cds1
E. Noguchi (2003)
Genome‐wide localization of pre‐RC sites and identification of replication origins in fission yeast
M. Hayashi (2007)
Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae.
Shawn J Szyjka (2005)
Rhp51(Rad51), and Rqh1 form an epistatic pathway required for the S-phase DNA damage checkpoint
N Willis (2009)
Microscopy techniques to examine DNA replication in fission yeast.
Marc D. Green (2009)
Mrc1 and Srs2
T. Robert (2006)
Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast
T. Nakamura (2004)
Autoinhibition and Autoactivation of the DNA Replication Checkpoint Kinase Cds1
Yong-jie Xu (2009)
Establishing the Program of Origin Firing during S Phase in Fission Yeast
P. Wu (2009)
Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast.
T. Takeda (2001)
Replication checkpoint enforced by kinases Cds1 and Chk1.
M. Boddy (1998)
Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival.
D. R. Williams (2005)
Mrc1 transduces signals of DNA replication stress to activate Rad53
A. Alcasabas (2001)
Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress.
M. Kai (2005)
Separate roles for the DNA damage checkpoint protein kinases in stabilizing DNA replication forks.
M. Segurado (2008)
Molecular genetics of Schizosaccharomyces pombe.
S. Sabatinos (2010)
Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex.
Marina N Nedelcheva (2005)
Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast.
Yong-jie Xu (2006)
A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication
M. Pacek (2004)
Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells
D. Jackson (1998)
Replication in Hydroxyurea: It's a Matter of Time
G. Alvino (2007)
Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1
Y. Zeng (1998)
The S / M checkpoint at 37°C and the recovery of viability of the mutant polδts3 require the crb2 +/rhp9 + gene in fission yeast
M. Grenon (1999)
Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase
O. Aparicio (1997)
Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint.
Benoît Froget (2008)
Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms.
K. Nestoras (2010)
The S/M checkpoint at 37 degrees C and the recovery of viability of the mutant poldeltats3 require the crb2+/rhp9+ gene in fission yeast.
M. Grenon (1999)
Mrc1 transduces
K. Furuya (2001)
A kinase from fission yeast responsible for blocking mitosis in S phase
H. Murakami (1995)
Cds1 Phosphorylation by Rad3-Rad26 Kinase Is Mediated by Forkhead-associated Domain Interaction with Mrc1*
K. Tanaka (2004)
Mrc1 protects uncapped budding yeast telomeres from exonuclease EXO1
A. Tsolou (2007)
S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe.
H. Lindsay (1998)
Threonine-11, Phosphorylated by Rad3 and ATM In Vitro, Is Required for Activation of Fission Yeast Checkpoint Kinase Cds1
K. Tanaka (2001)
Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint.
H. Lou (2008)
Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.
J. Cobb (2005)
dNTP pools determine fork progression and origin usage under replication stress
Jérôme Poli (2012)
Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53.
Alexander J. Osborn (2003)
The DNA replication checkpoint response stabilizes stalled replication forks
M. Lopes (2001)
Regulation of replication timing in fission yeast
S. M. Kim (2001)

This paper is referenced by
A visual atlas of meiotic protein dynamics in living fission yeast
Wilber Escorcia (2020)
Single-Molecule Studies of Replication Kinetics in Response to DNA Damage
Divya Ramalingam Iyer (2017)
A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M
Sriram Vijayraghavan (2016)
Preventing replication fork collapse to maintain genome integrity.
D. Cortez (2015)
Characterisation of unessential genes required for survival under conditions of DNA stress
Hassan Ahmed Ezzat (2020)
The extent of error-prone replication restart by homologous recombination is controlled by Exo1 and checkpoint proteins
E. Tsang (2014)
Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast
Silje Anda (2014)
FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.
G. Lossaint (2013)
Implementing super-resolution palm microscopy in fission yeast
Helen Armes (2017)
Characterization of the functions of Upf1 in the nucleus of Schizosaccharomyces pombe
Jianming Wang (2016)
The progress in the mechanistic studies of maintaining replication fork stability in eukaryotic cells
L. Yan (2014)
Chronic DNA Replication Stress Reduces Replicative Lifespan of Cells by TRP53-Dependent, microRNA-Assisted MCM2-7 Downregulation
Gongshi Bai (2016)
Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant
S. M. Kim (2020)
Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast
S. Sabatinos (2015)
The progress in the mechanistic studies of maintaining replication fork stability in eukaryotic cells
L. Yang (2014)
Essential Domains of Schizosaccharomyces pombe Rad8 Required for DNA Damage Response
L. Ding (2014)
Destabilization of the replication fork protection complex disrupts meiotic chromosome segregation
Wilber Escorcia (2017)
Fission yeast strains with circular chromosomes require the 9-1-1 checkpoint complex for the viability in response to the anti-cancer drug 5-fluorodeoxyuridine
H. M. Shamim (2017)
Cdc45 is limiting for replication initiation in humans
C. Köhler (2016)
Regulation of DNA-end resection at DNA double strand breaks and stalled replication forks
M. Villa (2018)
Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.
Yong-jie Xu (2016)
Replication fork stalling elicits chromatin compaction for the stability of stalling replication forks
G. Feng (2019)
Rad9/53BP1 protects stalled replication forks from degradation in Mec1/ATR‐defective cells
Matteo Villa (2018)
Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA
D. Iyer (2017)
The causes of replication stress and their consequences on genome stability and cell fate.
I. Magdalou (2014)
Translesion synthesis polymerases contribute to meiotic chromosome segregation and cohesin dynamics in Schizosaccharomyces pombe
T. Mastro (2020)
Rad4 Mainly Functions in Chk1-Mediated DNA Damage Checkpoint Pathway as a Scaffold Protein in the Fission Yeast Schizosaccharomyces pombe
M. Yue (2014)
Characterization of a Novel MMS-Sensitive Allele of Schizosaccharomyces pombe mcm4+
Nimna S. Ranatunga (2016)
Dynamics of DNA replication in a eukaryotic cell
T. Kelly (2019)
Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks
Sujan Devbhandari (2020)
Substrate Specificity of SAMHD1 Triphosphohydrolase Activity Is Controlled by Deoxyribonucleoside Triphosphates and Phosphorylation at Thr592.
Sunbok Jang (2016)
Increased Meiotic Crossovers and Reduced Genome Stability in Absence of Schizosaccharomyces pombe Rad16 (XPF)
T. Mastro (2014)
See more
Semantic Scholar Logo Some data provided by SemanticScholar