scholarly journals Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

2020 ◽  
Author(s):  
Mark C. Johnson ◽  
Geylani Can ◽  
Miguel Santos ◽  
Diana Alexander ◽  
Philip Zegerman
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
S Phase ◽  
Dna Lesions ◽  
Replication Initiation ◽  
Origin Firing ◽  
Initiation Factors ◽  
Dependent Inhibition ◽  
Rad53 Kinase ◽  
S Phase Checkpoint

AbstractAcross eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the ‘S-phase checkpoint’ with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.

scholarly journals Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mark C Johnson ◽  
Geylani Can ◽  
Miguel Monteiro Santos ◽  
Diana Alexander ◽  
Philip Zegerman
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Gene Amplification ◽  
S Phase ◽  
Checkpoint Inhibition ◽  
Origin Firing ◽  
Checkpoint Response ◽  
Initiation Factors ◽  
Dependent Inhibition ◽  
S Phase Checkpoint

Checkpoints maintain the order of cell cycle events during DNA damage or incomplete replication. How the checkpoint response is tailored to different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in budding yeast occurs by Rad53-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed, we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M, preventing gene amplification. In addition, we show that inhibition of Sld3 and Dbf4 in G1 prevents premature initiation at all origins at the G1/S transition. This study redefines the scope of the 'S-phase checkpoint' with implications for understanding checkpoint function in cancers that lack cell cycle controls.

scholarly journals Topoisomerase III Acts Upstream of Rad53p in the S-Phase DNA Damage Checkpoint

2001 ◽  
Vol 21 (21) ◽  
pp. 7150-7162 ◽  
Author(s):  
Ronjon K. Chakraverty ◽  
Jonathan M. Kearsey ◽  
Thomas J. Oakley ◽  
Muriel Grenon ◽  
Maria-Angeles de la Torre Ruiz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cellular Response ◽  
Cell Cycle Progression ◽  
Uv Light ◽  
S Phase ◽  
Dna Damaging Agents ◽  
Topoisomerase Iii ◽  
Rad53 Kinase ◽  
S Phase Checkpoint

ABSTRACT Deletion of the Saccharomyces cerevisiae TOP3gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391–8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Δ strains. We show that top3Δ mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion,top3Δ strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Δ mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.

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 Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

scholarly journals Critical Role for Mouse Hus1 in an S-Phase DNA Damage Cell Cycle Checkpoint

2003 ◽  
Vol 23 (3) ◽  
pp. 791-803 ◽  
Author(s):  
Robert S. Weiss ◽  
Philip Leder ◽  
Cyrus Vaziri
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Critical Role ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Null Mouse ◽  
Cycle Checkpoint ◽  
S Phase Checkpoint

ABSTRACT Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1− fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G2/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

scholarly journals A Fission Yeast Gene,him1+/dfp1+, Encoding a Regulatory Subunit for Hsk1 Kinase, Plays Essential Roles in S-Phase Initiation as Well as in S-Phase Checkpoint Control and Recovery from DNA Damage

1999 ◽  
Vol 19 (8) ◽  
pp. 5535-5547 ◽  
Author(s):  
Tadayuki Takeda ◽  
Keiko Ogino ◽  
Etsuko Matsui ◽  
Min Kwan Cho ◽  
Hiroyuki Kumagai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Fission Yeast ◽  
Kinase Activity ◽  
S Phase ◽  
Regulatory Subunit ◽  
Checkpoint Control ◽  
Regulatory Subunits ◽  
Hydroxyurea Treatment ◽  
S Phase Checkpoint

ABSTRACT Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G1/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1+ from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1+ is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G1. The protein level is low at START in G1, increases at the G1/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G1/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1+ is identical to rad35+ . Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

scholarly journals S-Phase Checkpoint Pathways Stimulate the Mobility of the Retrovirus-Like Transposon Ty1

2007 ◽  
Vol 27 (24) ◽  
pp. 8874-8885 ◽  
Author(s):  
M. Joan Curcio ◽  
Alison E. Kenny ◽  
Sharon Moore ◽  
David J. Garfinkel ◽  
Matthew Weintraub ◽  
...  
Keyword(s):  
Dna Damage ◽  
Reverse Transcriptase ◽  
Reverse Transcriptase Activity ◽  
S Phase ◽  
Dna Lesions ◽  
Restriction Factors ◽  
Transcriptase Activity ◽  
Checkpoint Pathway ◽  
Ty1 Mobility ◽  
S Phase Checkpoint

ABSTRACT The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101Δ mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101Δ mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

scholarly journals Visualization of Altered Replication Dynamics after DNA Damage in Human Cells

2004 ◽  
Vol 279 (19) ◽  
pp. 20067-20075 ◽  
Author(s):  
Catherine J. Merrick ◽  
Dean Jackson ◽  
John F. X. Diffley
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Replication Fork ◽  
S Phase ◽  
High Rate ◽  
Yeast Cells ◽  
Origin Firing ◽  
Large Numbers ◽  
Dependent Inhibition ◽  
Fork Stalling

Eukaryotic cells respond to DNA damage within the S phase by activating an intra-S checkpoint: a response that includes reducing the rate of DNA synthesis. In yeast cells this can occur via checkpoint-dependent inhibition of origin firing and stabilization of ongoing forks, together with a checkpoint-independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. We have developed strategies based on DNA fiber labeling that allow the quantitative assessment of rates of replication fork movement, origin firing, and fork stalling throughout the genome by examining large numbers of individually labeled replication forks. We show that exposing S phase cells to ionizing radiation induces a transient block to origin firing but does not affect fork rate or fork stalling. Alkylation damage by methyl methane sulfonate causes a slowing of fork movement and a high rate of fork stalling, in addition to inducing a block to new origin firing. Nucleotide depletion by hydroxyurea also reduces replication fork rate and increases stalling; moreover, in contrast to a recent report, we show that hydroxyurea induces a strong block to new origin firing. The DNA fiber labeling strategy provides a powerful new approach to analyze the dynamics of DNA replication in a perturbed S phase.

scholarly journals Phosphorylation of FANCD2 on Two Novel Sites Is Required for Mitomycin C Resistance

2006 ◽  
Vol 26 (18) ◽  
pp. 7005-7015 ◽  
Author(s):  
Gary P. H. Ho ◽  
Steven Margossian ◽  
Toshiyasu Taniguchi ◽  
Alan D. D'Andrea
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Cross Linking ◽  
Dependent Manner ◽  
Functional Connection ◽  
Cellular Resistance ◽  
Checkpoint Kinase ◽  
S Phase Checkpoint

ABSTRACT The Fanconi anemia (FA) pathway is a DNA damage-activated signaling pathway which regulates cellular resistance to DNA cross-linking agents. Cloned FA genes and proteins cooperate in this pathway, and monoubiquitination of FANCD2 is a critical downstream event. The cell cycle checkpoint kinase ATR is required for the efficient monoubiquitination of FANCD2, while another checkpoint kinase, ATM, directly phosphorylates FANCD2 and controls the ionizing radiation (IR)-inducible intra-S-phase checkpoint. In the present study, we identify two novel DNA damage-inducible phosphorylation sites on FANCD2, threonine 691 and serine 717. ATR phosphorylates FANCD2 on these two sites, thereby promoting FANCD2 monoubiquitination and enhancing cellular resistance to DNA cross-linking agents. Phosphorylation of the sites is required for establishment of the intra-S-phase checkpoint response. IR-inducible phosphorylation of threonine 691 and serine 717 is also dependent on ATM and is more strongly impaired when both ATM and ATR are knocked down. Threonine 691 is phosphorylated during normal S-phase progression in an ATM-dependent manner. These findings further support the functional connection of ATM/ATR kinases and FANCD2 in the DNA damage response and support a role for the FA pathway in the coordination of the S phase of the cell cycle.

The Role of MLL in DNA Damage Checkpoint and Its Implication in Leukemogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5332-5332
Author(s):  
Han Liu ◽  
Todd D Westergard ◽  
David Y Chen ◽  
Emily H.-Y. Cheng ◽  
James J.-D. Hsieh
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Critical Role ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Chromosome Band ◽  
Human Leukemia ◽  
Hox Gene ◽  
S Phase Checkpoint

Abstract Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which results in human cancers. Hence, it is of great significance to discover and investigate novel key constituents of the mammalian DNA damage response network. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a transcription co-activator that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathway must be involved, which remains to be discovered. Our recent studies demonstrated an intimate relationship between MLL and the cell cycle(Takeda et al. 2006, Genes & Development, 20, 2397–2409; Liu et al. 2007, Genes & Development, 21, 2385–2398). More importantly, our studies uncovered a critical role of MLL in executing the S phase checkpoint. We showed: Over-expression of MLL induces an S phase block. MLL accumulates in the S phase upon DNA damage. MLL deficiency results in radioresistant DNA synthesis (RDS) and chromatid-type chromosomal abnormalities, two signature characteristics of S phase checkpoint defects. We further determined the underlying mechanisms concerning the DNA damage-induced MLL accumulation. Our data showed that MLL is phosphorylated after DNA damage, which in turn blocks its degradation by SCFSkp2 in the S phase and results in the ultimate accumulation. Our data revealed the link between MLL and the S phase checkpoint, which provides novel insights into the mammalian cell cycle checkpoint network and human leukemia pathogenesis. Future studies utilizing murine leukemia models will be performed to examine whether MLL translocation compromises the S phase checkpoint and if the resulted dysfunction contributes to MLL leukemogenesis.

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