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

2001 ◽  
Vol 21 (15) ◽  
pp. 4938-4948 ◽  
Author(s):  
Yangzhou Wang ◽  
Marija Vujcic ◽  
David Kowalski
Keyword(s):  
Dna Replication ◽  
Mating Type ◽  
Replication Fork ◽  
Budding Yeast ◽  
Transcriptional Silencing ◽  
Replication Initiation ◽  
Replication Origins ◽  
Autonomously Replicating Sequence ◽  
Type Locus ◽  
Pause Site

ABSTRACT Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 andARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- andtrans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HMLARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival atHML led to the elimination of the pause sites at theHML ARS cluster and at the copy of ARS305inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed.

scholarly journals Activation of Silent Replication Origins at Autonomously Replicating Sequence Elements near the HML Locus in Budding Yeast

1999 ◽  
Vol 19 (9) ◽  
pp. 6098-6109 ◽  
Author(s):  
Marija Vujcic ◽  
Charles A. Miller ◽  
David Kowalski
Keyword(s):  
Replication Origin ◽  
Budding Yeast ◽  
Transcriptional Silencing ◽  
Growth Conditions ◽  
Replication Origins ◽  
Additional Time ◽  
Autonomously Replicating Sequence ◽  
Ars Elements ◽  
Active Replication ◽  
Chromosome Iii

ABSTRACT In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides betweenHML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303and in the process discovered a new ARS, ARS320. BothARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster withARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene,trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), orcis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for theHML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share keycis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at theHML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances fromHML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

scholarly journals A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase

2017 ◽  
Vol 292 (8) ◽  
pp. 3062-3073 ◽  
Author(s):  
Irina Bruck ◽  
Nalini Dhingra ◽  
Daniel L. Kaplan
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Budding Yeast ◽  
S Phase ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Minichromosome Maintenance ◽  
Initiation Factors ◽  
Complex Protein ◽  
Amplification Mechanism

The assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2–7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2–7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ϵ, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. We show here that Dpb11 stimulates DDK phosphorylation of the minichromosome maintenance complex protein Mcm4 alone and also of the Mcm2–7 complex and the dsDNA-loaded Mcm2–7 complex. We further demonstrate that Dpb11 can directly recruit DDK to Mcm4. A DDK phosphomimetic mutant of Mcm4 bound Dpb11 with substantially higher affinity than wild-type Mcm4, suggesting a mechanism to recruit Dpb11 to DDK-phosphorylated Mcm2–7. Furthermore, dsDNA-loaded Mcm2–7 harboring the DDK phosphomimetic Mcm4 mutant bound GINS in the presence of Dpb11, suggesting a mechanism for how GINS is recruited to Mcm2–7. We isolated a mutant of Dpb11 that is specifically defective for binding to Mcm4. This mutant, when expressed in budding yeast, diminished cell growth and DNA replication, substantially decreased Mcm4 phosphorylation, and decreased association of GINS with replication origins. We conclude that Dpb11 functions with DDK and Mcm4 in a positive amplification mechanism to trigger the assembly of the replication fork helicase.

scholarly journals Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

2018 ◽  
Author(s):  
Sarina Y. Porcella ◽  
Natasha C. Koussa ◽  
Colin P. Tang ◽  
Daphne N. Kramer ◽  
Priyanka Srivastava ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Leading Strand ◽  
Strand Initiation ◽  
Lagging Strand

AbstractDuring eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.Author summaryHalf of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

scholarly journals Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

2010 ◽  
Vol 30 (6) ◽  
pp. 1382-1396 ◽  
Author(s):  
Saravanabhavan Thangavel ◽  
Ramiro Mendoza-Maldonado ◽  
Erika Tissino ◽  
Julia M. Sidorova ◽  
Jinhu Yin ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Recq Helicases ◽  
Dna Replication Initiation ◽  
Replication Fork Progression ◽  
A Cell

ABSTRACT Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G1, after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.

scholarly journals A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 3261-3271 ◽  
Author(s):  
A M Merchant ◽  
Y Kawasaki ◽  
Y Chen ◽  
M Lei ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Replication Origins ◽  
Minichromosome Maintenance ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Dna Replication Initiation ◽  
Initiation Factors

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

scholarly journals Completion of Replication Map of Saccharomyces cerevisiae Chromosome III

2001 ◽  
Vol 12 (11) ◽  
pp. 3317-3327 ◽  
Author(s):  
Arkadi Poloumienko ◽  
Ann Dershowitz ◽  
Jitakshi De ◽  
Carol S. Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Complete Analysis ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Eukaryotic Chromosome ◽  
Ars Elements ◽  
Chromosome Iii

In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ∼40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements onS. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in ≤10% of cells in the population and two ARS elements active in ≥90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome.

Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins

1991 ◽  
Vol 11 (10) ◽  
pp. 5346-5355
Author(s):  
D D Dubey ◽  
L R Davis ◽  
S A Greenfeder ◽  
L Y Ong ◽  
J G Zhu ◽  
...  
Keyword(s):  
Mating Type ◽  
Transcriptional Repression ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Type Locus ◽  
Ars Elements ◽  
Dna Fragment ◽  
Dna Replication Origins ◽  
Chromosome Iii

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.

scholarly journals The interaction networks of the budding yeast and human DNA replication-initiation proteins

Cell Cycle ◽  
2019 ◽  
Vol 18 (6-7) ◽  
pp. 723-741 ◽  
Author(s):  
Rentian Wu ◽  
Aftab Amin ◽  
Ziyi Wang ◽  
Yining Huang ◽  
Marco Man-Hei Cheung ◽  
...  
Keyword(s):  
Dna Replication ◽  
Budding Yeast ◽  
Replication Initiation ◽  
Interaction Networks ◽  
Dna Replication Initiation ◽  
Human Dna ◽  
Replication Initiation Proteins

scholarly journals Pervasive Phenotypic Impact of a Large Nonrecombining Introgressed Region in Yeast

2020 ◽  
Vol 37 (9) ◽  
pp. 2520-2530 ◽  
Author(s):  
Christian Brion ◽  
Claudia Caradec ◽  
David Pflieger ◽  
Anne Friedrich ◽  
Joseph Schacherer
Keyword(s):  
Mating Type ◽  
Budding Yeast ◽  
Natural Populations ◽  
Phenotypic Effect ◽  
Genetic Characteristics ◽  
Type Locus ◽  
First Time ◽  
The Relationship ◽  
Insight Into ◽  
Introgressed Region

Abstract To explore the origin of the diversity observed in natural populations, many studies have investigated the relationship between genotype and phenotype. In yeast species, especially in Saccharomyces cerevisiae, these studies are mainly conducted using recombinant offspring derived from two genetically diverse isolates, allowing to define the phenotypic effect of genetic variants. However, large genomic variants such as interspecies introgressions are usually overlooked even if they are known to modify the genotype–phenotype relationship. To have a better insight into the overall phenotypic impact of introgressions, we took advantage of the presence of a 1-Mb introgressed region, which lacks recombination and contains the mating-type determinant in the Lachancea kluyveri budding yeast. By performing linkage mapping analyses in this species, we identified a total of 89 loci affecting growth fitness in a large number of conditions and 2,187 loci affecting gene expression mostly grouped into two major hotspots, one being the introgressed region carrying the mating-type locus. Because of the absence of recombination, our results highlight the presence of a sexual dimorphism in a budding yeast for the first time. Overall, by describing the phenotype–genotype relationship in the Lachancea kluyveri species, we expanded our knowledge on how genetic characteristics of large introgression events can affect the phenotypic landscape.

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