DNA Replication Forks Pause at Silent Origins near the <i>HML</i> Locus in Budding Yeast

Wang, Yangzhou; Vujcic, Marija; Kowalski, David

doi:10.1128/mcb.21.15.4938-4948.2001

Cited by 34 publications

(31 citation statements)

References 85 publications

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“…Unusually large replicons may have an increased propensity for defects in replication fork progression due to normal fork impediments or limited processivity, leading to DNA damage. Hundreds of sites in the genome present obstacles or potential obstacles to fork progression, including tRNA genes, rDNA repeats, and unfired replication origins (7,14,41). A reduction in replication origin usage not only creates more potential pause sites at each unfired origin but also reduces the number of replication forks relative to the number of pause sites.…”

Section: Discussionmentioning

confidence: 99%

Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

Gibson

Aparicio

et al. 2004

Molecular and Cellular Biology

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Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5⌬ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5⌬ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5⌬ cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5⌬ cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.Duplication of eukaryotic chromosomes involves the initiation of DNA synthesis from multiple origins of replication distributed along each chromosome. Although chromosomal DNA replication is restricted to the S-phase period of the cell cycle, individual replication origins initiate DNA synthesis (or "fire") at different times during S phase in a regulated fashion such that each replication origin has a characteristic initiation time within S phase. The exact nature of this regulation is not fully understood but appears to be influenced by two apparently unrelated factors: the local chromatin environment of each origin, which establishes the relative order of origin firing prior to S phase (reviewed in reference 43); and checkpoint signaling pathways, which modulate the extent to which the initiation of certain origins is delayed, particularly in response to replication defects or DNA damage (21,29,31,33).The pre-replicative complex (pre-RC) assembles at and governs the function of each replication origin (reviewed in reference 4). Activation of the pre-RC results in its conversion into two, divergent, replication fork complexes (replisomes) through the recruitment of additional DNA synthesis factors, such as Cdc45 and DNA polymerases. Pre-RC activation requires the function of cyclin-dependent kinase (CDK), which consists of a catalytic subunit (Cdk1) controlled by a cyclin subunit whose expression and stability are cell cycle regulated. In Saccharomyces ce...

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Section: Discussionmentioning

confidence: 99%

Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

Gibson

Aparicio

et al. 2004

Molecular and Cellular Biology

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“…It is clear that replication forks pause frequently during a normal S phase (Deshpande and Newlon 1996;Wang et al 2001), often leading to parental strand breakage. Because these can generally be repaired by recombination with the adjacent sister chromatid (Myung et al 2001;Schär 2001), it would not, in most situations, be in the interest of the cell to provoke a cell-cycle arrest in response to these fork-associated events.…”

Section: A Cellular Mechanism Sensitive To Replication Fork Numbermentioning

confidence: 99%

ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase

Shimada¹,

Pasero²,

Gasser³

2002

Genes Dev.

209

199

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The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks. As cells proliferate, the genome must be replicated and segregated with high fidelity. Surveillance mechanisms, or checkpoints, detect damaged DNA or on-going replication and block cell cycle progression in order to allow adequate time to repair the damaged DNA or to complete replication (Weinert 1998;Zhou and Elledge 2000). Failure to delay the cell cycle can convert an easily reparable DNA lesion into one more deleterious, resulting in chromosomal rearrangements or loss. The importance of this mechanism is underscored by a large number of heritable human diseases that arise from defects in checkpoint or DNA-damage repair functions (for review, see Kastan 1997).Genetic analysis of the DNA damage checkpoint pathway in yeast has allowed classification of its components into sensors, which detect DNA damage, adaptors, which integrate and transmit the signal, and effector kinases, which promote downstream functions including the induction of repair genes, suppression of cell cycle progression, the arrest of replication polymerases, downregulation of late origins and of sister chromatid segregation (for review, see Zhou and Elledge 2000; Melo and Toczyski 2002). In G1 and G2 phases of the budding yeast cell cycle, a DNA-bound complex of Rad17/Mec3/ Ddc1 and Rad24 proteins, as well as a complex containing the ATM homolog Mec1p and its cofactor Ddc2p, play crucial roles as sensors. These communicate through Rad9p to activate the major effector kinases, Rad53p (CHK2 in other species) and Chk1p. These components and their roles are conserved from yeast to man (Melo and Toczyski 2002).The response of the cell to DNA damage or fork arrest during S phase is distinct from the G1/S or G2/M checkpoints, both at the level of sensors and with respect to the cellular response to Rad53p activation. It is useful to consider this intra-S-phase response with respect to two operationally distinct lesions. The first arises from replication forks stalled by high concentra...

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“…Fork progression can be compromised by exogenous stress, such as DNA damage or inhibitors of DNA replication (16,37). Blocking (3,21) and transient pausing (11,17,18,44) of replication fork progression during the normal process of chromosome replication at sites where nonnucleosomal proteins are tightly bound to DNA are well documented. Interestingly, in Schizosaccharomyces pombe, recombination has been shown to be a requirement in promoting cell viability when fork progression is blocked (23).…”

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confidence: 99%

Replication Fork Progression Is Impaired by Transcription in Hyperrecombinant Yeast Cells Lacking a Functional THO Complex

Wellinger

Prado

Aguilera

2006

Molecular and Cellular Biology

151

121

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THO/TREX is a conserved, eukaryotic protein complex operating at the interface between transcription and messenger ribonucleoprotein (mRNP) metabolism. THO mutations impair transcription and lead to increased transcription-associated recombination (TAR). These phenotypes are dependent on the nascent mRNA; however, the molecular mechanism by which impaired mRNP biogenesis triggers recombination in THO/TREX mutants is unknown. In this study, we provide evidence that deficient mRNP biogenesis causes slowdown or pausing of the replication fork in hpr1⌬ mutants. Impaired replication appears to depend on sequence-specific features since it was observed upon activation of lacZ but not leu2 transcription. Replication fork progression could be partially restored by hammerhead ribozyme-guided self-cleavage of the nascent mRNA. Additionally, hpr1⌬ increased the number of S-phase but not G 2 -dependent TAR events as well as the number of budded cells containing Rad52 repair foci. Our results link transcription-dependent genomic instability in THO mutants with impaired replication fork progression, suggesting a molecular basis for a connection between inefficient mRNP biogenesis and genetic instability.Genetic instability of a DNA fragment can be induced by transcription, a phenomenon referred to as transcription-associated recombination (TAR). Recombination has been shown to increase in actively transcribed genes in bacteria, yeasts, and humans (1). This is the case for RNA polymerase II (RNAPII)-dependent transcription, as first shown for yeast (43). Despite the ubiquity and relevance of TAR, its mechanism(s) remains unknown. Transcription-dependent hyperrecombination is a hallmark phenotype of mutants of the THO complex in the yeast S. cerevisiae (4,36). THO is a conserved, eukaryotic multiprotein complex, containing Hpr1, Mft1, Tho2, and Thp2 in yeast (5). Moreover, THO acts at the interface between transcription and mRNP export via its interaction with Sub2 and Yra1 in a highmolecular-weight complex termed TREX (19,40). THO/TREX components are recruited to an active gene during transcription elongation. Hpr1 directly interacts with Sub2 and facilitates the binding of Sub2 and Yra1 to nascent transcripts (46). Mutations in most factors involved in messenger RNP (mRNP) biogenesis and export, including Sub2, Yra1, Thp1-Sac3, Nab2, Mex67, and Mtr2, confer a gene expression defect and a transcription-dependent hyperrecombination phenotype comparable to that described for THO mutant strains (10, 19). The similarity of these phenotypes suggests that correct processing of a number of nuclear steps, leading to export-competent mRNP particles, is important in preventing transcription-dependent genomic instability (29). It remains to be seen whether TAR events stimulated in THO mutants result from the same mechanism(s) as spontaneous TAR events occurring in wild-type cells.DNA replication occurs during S phase of the cell cycle and is initiated at multiple origins of replication (20). Once established, a single replication fork wi...

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DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast

Cited by 34 publications

References 85 publications

Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

Diminished S-Phase Cyclin-Dependent Kinase Function Elicits Vital Rad53-Dependent Checkpoint Responses in Saccharomyces cerevisiae

ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase

Replication Fork Progression Is Impaired by Transcription in Hyperrecombinant Yeast Cells Lacking a Functional THO Complex

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