scholarly journals MCM2–7 Proteins Are Essential Components of Prereplicative Complexes that Accumulate Cooperatively in the Nucleus during G1-phase and Are Required to Establish, But Not Maintain, the S-phase Checkpoint

2001 ◽  
Vol 12 (11) ◽  
pp. 3658-3667 ◽  
Author(s):  
Karim Labib ◽  
Stephen E. Kearsey ◽  
John F.X. Diffley
Keyword(s):  
Chromosome Replication ◽  
S Phase ◽  
G1 Phase ◽  
Checkpoint Control ◽  
Direct Role ◽  
Essential Components ◽  
Mcm Proteins ◽  
Prereplicative Complex ◽  
Genomic Footprinting ◽  
Checkpoint Signal

A prereplicative complex (pre-RC) of proteins is assembled at budding yeast origins of DNA replication during the G1-phase of the cell cycle, as shown by genomic footprinting. The proteins responsible for this prereplicative footprint have yet to be identified but are likely to be involved in the earliest stages of the initiation step of chromosome replication. Here we show that MCM2–7 proteins are essential for both the formation and maintenance of the pre-RC footprint at the origin ARS305. It is likely that pre-RCs contain heteromeric complexes of MCM2–7 proteins, since degradation of Mcm2, 3, 6, or 7 during G1-phase, after pre-RC formation, causes loss of Mcm4 from the nucleus. It has been suggested that pre-RCs on unreplicated chromatin may generate a checkpoint signal that inhibits premature mitosis during S-phase. We show that, although mitosis does indeed occur in the absence of replication if MCM proteins are degraded during G1-phase, anaphase is prevented if MCMs are degraded during S-phase. Our data indicate that pre-RCs do not play a direct role in checkpoint control during chromosome replication.

scholarly journals Divergent S Phase Checkpoint Activation Arising from Prereplicative Complex Deficiency Controls Cell Survival

2009 ◽  
Vol 20 (17) ◽  
pp. 3953-3964 ◽  
Author(s):  
Eric Lau ◽  
Gary G. Chiang ◽  
Robert T. Abraham ◽  
Wei Jiang
Keyword(s):  
Dna Replication ◽  
Cancer Cells ◽  
S Phase ◽  
Checkpoint Control ◽  
Multiple Cancer ◽  
Checkpoint Activation ◽  
Diploid Cells ◽  
Prereplicative Complex ◽  
Stress Damage ◽  
S Phase Checkpoint

The DNA replication machinery plays additional roles in S phase checkpoint control, although the identities of the replication proteins involved in checkpoint activation remain elusive. Here, we report that depletion of the prereplicative complex (pre-RC) protein Cdc6 causes human nontransformed diploid cells to arrest nonlethally in G1-G1/S and S phase, whereas multiple cancer cell lines undergo G1-G1/S arrest and cell death. These divergent phenotypes are dependent on the activation, or lack thereof, of an ataxia telangiectasia and Rad3-related (ATR)-dependent S phase checkpoint that inhibits replication fork progression. Although pre-RC deficiency induces chromatin structural alterations in both nontransformed and cancer cells that normally lead to ATR checkpoint activation, the sensor mechanisms in cancer cells seem to be compromised such that higher levels of DNA replication stress/damage are required to trigger checkpoint response. Our results suggest that therapy-induced disruption of pre-RC function might exert selective cytotoxic effects on tumor cells in human patients.

DNA polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast

1995 ◽  
Vol 108 (9) ◽  
pp. 3109-3118 ◽  
Author(s):  
G. D'Urso ◽  
B. Grallert ◽  
P. Nurse
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
S Phase ◽  
Replication Initiation ◽  
Direct Immunofluorescence ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Initiation Complex ◽  
Dna Polymerase Alpha ◽  
Checkpoint Signal

Genetic analysis in the yeast Schizosaccharomyces pombe has shown that three genes cdc18, cut5, and cdt1, are essential for DNA synthesis and also for the checkpoint control that couples completion of DNA replication to the onset of mitosis. To test whether assembly of the replication initiation complex is an important element in the checkpoint control pathway we have investigated if DNA polymerase alpha (pol1), a component of the initiation complex, is essential for the S-phase checkpoint control. We show that germinating S. pombe spores disrupted for the pol1 gene enter mitosis despite defects in DNA synthesis. This is shown by monitoring septation index, DNA content, and by direct immunofluorescence of mitotic spindles using antibodies to alpha-tubulin. In addition we have isolated six temperature sensitive mutants in the pol1 gene that cause cell cycle arrest when grown at the nonpermissive temperature. Our experiments support a model in which DNA polymerase alpha, in addition to being part of the initiation complex, is required for a checkpoint signal that is activated as cells traverse START, and is essential to prevent mitosis until S phase has been completed. In contrast, proteins responsible for the elongation of DNA may not be necessary for this checkpoint signal.

scholarly journals Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

2011 ◽  
Vol 192 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Marjorie A. Kuipers ◽  
Timothy J. Stasevich ◽  
Takayo Sasaki ◽  
Korey A. Wilson ◽  
Kristin L. Hazelwood ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Replication Fork ◽  
S Phase ◽  
G1 Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycles ◽  
Replication Arrest ◽  
Minichromosome Maintenance Protein ◽  
Mcm Proteins

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.

scholarly journals Mcm2, but Not Rpa, Is a Component of the Mammalian Early G1-Phase Prereplication Complex

1999 ◽  
Vol 146 (4) ◽  
pp. 709-722 ◽  
Author(s):  
Daniela S. Dimitrova ◽  
Ivan T. Todorov ◽  
Thomas Melendy ◽  
David M. Gilbert
Keyword(s):  
Chinese Hamster ◽  
S Phase ◽  
G1 Phase ◽  
Replication Forks ◽  
Active Replication ◽  
Egg Extracts ◽  
Late Telophase ◽  
Mcm Proteins ◽  
Xenopus Egg Extracts ◽  
Stepwise Assembly

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1–mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.

scholarly journals Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 550
Author(s):  
Indra A. Shaltiel ◽  
Alba Llopis ◽  
Melinda Aprelia ◽  
Rob Klompmaker ◽  
Apostolos Menegakis ◽  
...  
Keyword(s):  
Dna Damage ◽  
Normal Cell ◽  
S Phase ◽  
G1 Phase ◽  
Anaphase Promoting Complex ◽  
Inhibitory Effects ◽  
Pocket Proteins ◽  
Cyclin Dependent Kinases ◽  
Independent Functions ◽  
Phase Entry

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.

scholarly journals Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
Author(s):  
Xuan Wang ◽  
Grzegorz Ira ◽  
José Antonio Tercero ◽  
Allyson M. Holmes ◽  
John F. X. Diffley ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Dna Ligases ◽  
Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

scholarly journals Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

1975 ◽  
Vol 66 (1) ◽  
pp. 95-101 ◽  
Author(s):  
K D Ley
Keyword(s):  
Time Course ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Protein S ◽  
S Phase ◽  
Supernatant Fraction ◽  
G1 Phase ◽  
Differential Centrifugation ◽  
Chinese Hamster Cells ◽  
Soluble Supernatant

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.

scholarly journals Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling

2006 ◽  
Vol 173 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Shan Yan ◽  
Howard D. Lindsay ◽  
W. Matthew Michael
Keyword(s):  
Sperm Chromatin ◽  
Checkpoint Control ◽  
Direct Role ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Oligonucleotide Duplex ◽  
Stalled Replication Forks

TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3–related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.

scholarly journals CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

1999 ◽  
Vol 340 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Parisa DANAIE ◽  
Michael ALTMANN ◽  
Michael N. HALL ◽  
Hans TRACHSEL ◽  
Stephen B. HELLIWELL
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
G1 Phase ◽  
Mrna Levels ◽  
Wild Type ◽  
Phase Progression ◽  
Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

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