scholarly journals Competition for MCM Loading at Origins Establishes Replication Timing Patterns

2020 ◽  
Author(s):  
Livio Dukaj ◽  
Nicholas Rhind
Keyword(s):  
Stress Reduction ◽  
Cell Cycle Progression ◽  
Origin Recognition Complex ◽  
Optimal Growth ◽  
Replication Timing ◽  
Growth Conditions ◽  
S Phase ◽  
Yeast Cells ◽  
Origins Of Replication ◽  
Increased Sensitivity

AbstractLoading of the MCM replicative helicase onto origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The number of MCM loaded on origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide characteristics of MCM loading and their direct effect on replication timing remain unclear. In order to probe MCM loading dynamics and its effect on replication timing, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their initiation during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels not limiting for MCM loading. Our results support a model in which the loading activity of origins, controlled by their ability to recruit ORC and compete for MCM, determines the number of helicases loaded, which in turn affects replication timing.

scholarly journals The capacity of origins to load mcm establishes replication timing patterns

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009467
Author(s):  
Livio Dukaj ◽  
Nicholas Rhind
Keyword(s):  
Stress Reduction ◽  
Cell Cycle Progression ◽  
Origin Recognition Complex ◽  
Optimal Growth ◽  
Replication Timing ◽  
Growth Conditions ◽  
S Phase ◽  
Yeast Cells ◽  
Origins Of Replication ◽  
Primary Determinant

Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins’ ability to recruit ORC and compete for MCM when MCM becomes limiting.

Analysis of mutant origin recognition complex with reduced ATPase activity in vivo and in vitro

2008 ◽  
Vol 413 (3) ◽  
pp. 535-543 ◽  
Author(s):  
Masaya Takehara ◽  
Masaki Makise ◽  
Hitomi Takenaka ◽  
Teita Asano ◽  
Tohru Mizushima
Keyword(s):  
Atpase Activity ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
Aaa Atpases ◽  
Origin Recognition

In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA+ (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Orc1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA+ proteins. Plasmid-shuffling analysis revealed that Asn600, Arg694 and Arg704 are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg694→Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg694 of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.

scholarly journals A Forkhead Transcription Factor Is Important for True Hyphal as well as Yeast Morphogenesis in Candida albicans

2002 ◽  
Vol 1 (5) ◽  
pp. 787-798 ◽  
Author(s):  
Eric S. Bensen ◽  
Scott G. Filler ◽  
Judith Berman
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Hyphal Growth ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Cycle Progression ◽  
Forkhead Transcription Factors

ABSTRACT Candida albicans is an important pathogen of immunocompromised patients which grows with true hyphal, pseudohyphal, and yeast morphologies. The dynamics of cell cycle progression are markedly different in true hyphal relative to pseudohyphal and yeast cells, including nuclear movement and septin ring positioning. In Saccharomyces cerevisiae, two forkhead transcription factors (ScFKH1 and ScFKH2) regulate the expression of B-cyclin genes. In both S. cerevisiae and Schizosaccharomyces pombe, forkhead transcription factors also influence morphogenesis. To explore the molecular mechanisms that connect C. albicans morphogenesis with cell cycle progression, we analyzed CaFKH2, the single homolog of S. cerevisiae FKH1/FKH2. C. albicans cells lacking CaFkh2p formed constitutive pseudohyphae under all yeast and hyphal growth conditions tested. Under hyphal growth conditions levels of hyphae-specific mRNAs were reduced, and under yeast growth conditions levels of several genes encoding proteins likely to be important for cell wall separation were reduced. Together these results imply that Fkh2p is required for the morphogenesis of true hyphal as well as yeast cells. Efg1p and Cph1p, two transcription factors that contribute to C. albicans hyphal growth, were not required for the pseudohyphal morphology of fkh2 mutants, implying that Fkh2p acts in pathways downstream of and/or parallel to Efg1p and Cph1p. In addition, cells lacking Fkh2p were unable to damage human epithelial or endothelial cells in vitro, suggesting that Fkh2p contributes to C. albicans virulence.

scholarly journals Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells

2007 ◽  
Vol 402 (2) ◽  
pp. 397-403 ◽  
Author(s):  
Masaki Makise ◽  
Naoko Takahashi ◽  
Kazuya Matsuda ◽  
Fumiko Yamairi ◽  
Keitarou Suzuki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
High Temperatures ◽  
Cell Cycle Progression ◽  
Origin Recognition Complex ◽  
Terminal Region ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
Growth Defects ◽  
Cycle Arrest ◽  
Origin Recognition

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 °C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469–8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.

scholarly journals A Role for the Replication Proteins PCNA, RF-C, Polymerase ε and Cdc45 in Transcriptional Silencing in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (3) ◽  
pp. 1171-1182
Author(s):  
Ann E Ehrenhofer-Murray ◽  
Rohinton T Kamakaka ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Origin Recognition Complex ◽  
Transcriptional Silencing ◽  
Replication Timing ◽  
Replication Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycle Progression

Abstract Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase ε (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed.

scholarly journals Cell Cycle Progression in G1 and S Phases Is CCR4 Dependent following Ionizing Radiation or Replication Stress in Saccharomyces cerevisiae

2004 ◽  
Vol 3 (2) ◽  
pp. 430-446 ◽  
Author(s):  
Tammy J. Westmoreland ◽  
Jeffrey R. Marks ◽  
John A. Olson ◽  
Eric M. Thompson ◽  
Michael A. Resnick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Ionizing Radiation ◽  
Cell Cycle Progression ◽  
Replication Stress ◽  
S Phase ◽  
Checkpoint Control ◽  
New Genes ◽  
Increased Sensitivity ◽  
Transcription Complexes

ABSTRACT To identify new nonessential genes that affect genome integrity, we completed a screening for diploid mutant Saccharomyces cerevisiae strains that are sensitive to ionizing radiation (IR) and found 62 new genes that confer resistance. Along with those previously reported (Bennett et al., Nat. Genet. 29:426-434, 2001), these genes bring to 169 the total number of new IR resistance genes identified. Through the use of existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4-NOT and RNA polymerase-associated factor 1 (PAF1)-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G1 cell population is radiosensitive. Consistent with a role in G1, diploid ccr4Δ cells irradiated in G1 show enhanced lethality compared to cells exposed as a synchronous G2 population. In addition, a prolonged RAD9-dependent G1 arrest occurred following IR of ccr4Δ cells and CCR4 is a member of the RAD9 epistasis group, thus confirming a role for CCR4 in checkpoint control. Moreover, ccr4Δ cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G2 followed by cellular lysis. This S-phase replication defect is separate from that seen for rad52 mutants, since rad52Δ ccr4Δ cells show increased sensitivity to HU compared to rad52Δ or ccr4Δ mutants alone. These results indicate that cell cycle transition through G1 and S phases is CCR4 dependent following radiation or replication stress.

scholarly journals Tolerance of Sir1p/Origin Recognition Complex-Dependent Silencing for Enhanced Origin Firing at HMRa

2006 ◽  
Vol 26 (5) ◽  
pp. 1955-1966 ◽  
Author(s):  
Kristopher H. McConnell ◽  
Philipp Müller ◽  
Catherine A. Fox
Keyword(s):  
Origin Recognition Complex ◽  
Substantial Reduction ◽  
Replication Timing ◽  
S Phase ◽  
Replication Initiation ◽  
Silent Chromatin ◽  
Replication Origins ◽  
Origin Firing ◽  
Sir Proteins ◽  
Origin Recognition

ABSTRACT The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMR a locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMR a. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMR a silencing, origin firing, and replication timing. Origin firing within HMR a and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMR a replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMR a quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.

scholarly journals Isolation of a gene required for programmed initiation of development by Aspergillus nidulans.

1992 ◽  
Vol 12 (9) ◽  
pp. 3827-3833 ◽  
Author(s):  
T H Adams ◽  
W A Hide ◽  
L N Yager ◽  
B N Lee
Keyword(s):  
Life Cycle ◽  
Aspergillus Nidulans ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Nutrient Deprivation ◽  
Deletion Mutants ◽  
Wild Type ◽  
Microbial Development ◽  
Type Strains ◽  
Posttranscriptional Level

In contrast to many other cases in microbial development, Aspergillus nidulans conidiophore production initiates primarily as a programmed part of the life cycle rather than as a response to nutrient deprivation. Mutations in the acoD locus result in "fluffy" colonies that appear to grow faster than the wild type and proliferate as undifferentiated masses of vegetative cells. We show that unlike wild-type strains, acoD deletion mutants are unable to make conidiophores under optimal growth conditions but can be induced to conidiate when growth is nutritionally limited. The requirement for acoD in conidiophore development occurs prior to activation of brlA, a primary regulator of development. The acoD transcript is present both in vegetative hyphae prior to developmental induction and in developing cultures. However, the effects of acoD mutations are detectable only after developmental induction. We propose that acoD activity is primarily controlled at the posttranscriptional level and that it is required to direct developmentally specific changes that bring about growth inhibition and activation of brlA expression to result in conidiophore development.

scholarly journals The Meiosis-Specific Crs1 Cyclin Is Required for Efficient S-Phase Progression and Stable Nuclear Architecture

2021 ◽  
Vol 22 (11) ◽  
pp. 5483
Author(s):  
Luisa F. Bustamante-Jaramillo ◽  
Celia Ramos ◽  
Cristina Martín-Castellanos
Keyword(s):  
Cell Cycle ◽  
Translational Control ◽  
Cell Cycle Progression ◽  
Nuclear Architecture ◽  
Strand Break ◽  
Spindle Pole ◽  
S Phase ◽  
Cycle Progression ◽  
Single Wave ◽  
Phase Progression

Cyclins and CDKs (Cyclin Dependent Kinases) are key players in the biology of eukaryotic cells, representing hubs for the orchestration of physiological conditions with cell cycle progression. Furthermore, as in the case of meiosis, cyclins and CDKs have acquired novel functions unrelated to this primal role in driving the division cycle. Meiosis is a specialized developmental program that ensures proper propagation of the genetic information to the next generation by the production of gametes with accurate chromosome content, and meiosis-specific cyclins are widespread in evolution. We have explored the diversification of CDK functions studying the meiosis-specific Crs1 cyclin in fission yeast. In addition to the reported role in DSB (Double Strand Break) formation, this cyclin is required for meiotic S-phase progression, a canonical role, and to maintain the architecture of the meiotic chromosomes. Crs1 localizes at the SPB (Spindle Pole Body) and is required to stabilize the cluster of telomeres at this location (bouquet configuration), as well as for normal SPB motion. In addition, Crs1 exhibits CDK(Cdc2)-dependent kinase activity in a biphasic manner during meiosis, in contrast to a single wave of protein expression, suggesting a post-translational control of its activity. Thus, Crs1 displays multiple functions, acting both in cell cycle progression and in several key meiosis-specific events.

Sign in / Sign up

Export Citation Format

Share Document