scholarly journals POLD replicates both strands of small kilobase-long replication bubbles initiated at a majority of human replication origins

2017 ◽  
Author(s):  
Artem V. Artemov ◽  
Maria A. Andrianova ◽  
Georgii A. Bazykin ◽  
Vladimir B. Seplyarskiy
Keyword(s):  
Dna Sequencing ◽  
Replication Fork ◽  
Additional Data ◽  
Replication Initiation ◽  
Mutational Bias ◽  
Replication Origins ◽  
Induced Mutations ◽  
Mutator Phenotype ◽  
New Model ◽  
Polymerase Epsilon

AbstractError-prone mutants of polymerase epsilon (POLE*) or polymerase delta (POLD1*) induce a mutator phenotype in human cancers. Here we show that the rate of mutations introduced by POLD1* is elevated by 50%, while the rate of POLE*-induced mutations is decreased twofold, within one kilobase from replication origins. These results support a model in which POLD1 replicates both the leading and the lagging strands within a kilobase from an origin. The magnitude of the mutational bias suggests that the probability of an individual origin to initiate replication exceeds 50%, which is much higher than previous estimates. Using additional data from nascent DNA sequencing and Okazaki fragments sequencing (OK-seq) experiments, we showed that a majority of origins are firing at each replication round, but the initiated replication fork does not propagate further than 1Kb in both directions. Analyses based on mutational data and on OK-seq data concordantly suggest that only approximately a quarter of fired origins result in a processive replication fork. Taken together, our results provide a new model of replication initiation.

scholarly journals The S-phase Cyclin Clb5 Promotes rDNA Stability by Maintaining Replication Initiation Efficiency in rDNA

2020 ◽  
Author(s):  
Mayuko Goto ◽  
Mariko Sasaki ◽  
Takehiko Kobayashi
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Tandem Repeats ◽  
Genome Instability ◽  
S Phase ◽  
Replication Initiation ◽  
Replication Origins ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replication Fork Arrest

ABSTRACTRegulation of replication origins is important for complete duplication of the genome, but the effect of origin activation on the cellular response to replication stress is poorly understood. The budding yeast ribosomal RNA gene (rDNA) forms tandem repeats and undergoes replication fork arrest at the replication fork barrier (RFB), inducing DNA double-strand breaks (DSBs) and genome instability accompanied by copy number alterations. Here we demonstrate that the S-phase cyclin Clb5 promotes rDNA stability. Absence of Clb5 led to reduced efficiency of replication initiation in rDNA but had little effect on the amount of replication forks arrested at the RFB, suggesting that arrival of the converging fork is delayed and forks are more stably arrested at the RFB. Deletion of CLB5 affected neither DSB formation nor its repair at the RFB, but led to an accumulation of recombination intermediates. Therefore, arrested forks at the RFB may be subject to DSB-independent, recombination-dependent rDNA instability. The rDNA instability in clb5Δ was not completely suppressed by the absence of Fob1, which is responsible for fork arrest at the RFB. Thus, Clb5 establishes the proper interval for active replication origins and shortens the travel distance for DNA polymerases, which may reduce Fob1-independent DNA damage.

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 The S-Phase Cyclin Clb5 Promotes rRNA Gene (rDNA) Stability by Maintaining Replication Initiation Efficiency in rDNA

2021 ◽  
Vol 41 (5) ◽  
Author(s):  
Mayuko Goto ◽  
Mariko Sasaki ◽  
Takehiko Kobayashi
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Tandem Repeats ◽  
Genome Instability ◽  
S Phase ◽  
Replication Initiation ◽  
Rrna Gene ◽  
Replication Origins ◽  
Dna Double Strand Breaks ◽  
Replication Fork Arrest

ABSTRACT Regulation of replication origins is important for complete duplication of the genome, but the effect of origin activation on the cellular response to replication stress is poorly understood. The budding yeast rRNA gene (rDNA) forms tandem repeats and undergoes replication fork arrest at the replication fork barrier (RFB), inducing DNA double-strand breaks (DSBs) and genome instability accompanied by copy number alterations. Here, we demonstrate that the S-phase cyclin Clb5 promotes rDNA stability. Absence of Clb5 led to reduced efficiency of replication initiation in rDNA but had little effect on the number of replication forks arrested at the RFB, suggesting that arrival of the converging fork is delayed and forks are more stably arrested at the RFB. Deletion of CLB5 affected neither DSB formation nor its repair at the RFB but led to homologous recombination-dependent rDNA instability. Therefore, arrested forks at the RFB may be subject to DSB-independent, recombination-dependent rDNA instability. The rDNA instability in clb5Δ was not completely suppressed by the absence of Fob1, which is responsible for fork arrest at the RFB. Thus, Clb5 establishes the proper interval for active replication origins and shortens the travel distance for DNA polymerases, which may reduce Fob1-independent DNA damage.

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 Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress

2008 ◽  
Vol 191 (2) ◽  
pp. 486-493 ◽  
Author(s):  
Adam M. Breier ◽  
Alan D. Grossman
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Initiation ◽  
Pass Through ◽  
Bacillus Subtilis Chromosome ◽  
Rapid Signaling ◽  
Replication Initiator

ABSTRACT DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.

scholarly journals Multiple replication origins with diverse control mechanisms in Haloarcula hispanica

2013 ◽  
Vol 42 (4) ◽  
pp. 2282-2294 ◽  
Author(s):  
Zhenfang Wu ◽  
Jingfang Liu ◽  
Haibo Yang ◽  
Hailong Liu ◽  
Hua Xiang
Keyword(s):  
Replication Initiation ◽  
Control Mechanisms ◽  
Replication Origins ◽  
Dna Mutation ◽  
Genome Wide ◽  
Marker Frequency ◽  
Haloarcula Hispanica ◽  
Specific Control ◽  
Main Chromosome ◽  
Multiple Replication

Abstract The use of multiple replication origins in archaea is not well understood. In particular, little is known about their specific control mechanisms. Here, we investigated the active replication origins in the three replicons of a halophilic archaeon, Haloarcula hispanica, by extensive gene deletion, DNA mutation and genome-wide marker frequency analyses. We revealed that individual origins are specifically dependent on their co-located cdc6 genes, and a single active origin/cdc6 pairing is essential and sufficient for each replicon. Notably, we demonstrated that the activities of oriC1 and oriC2, the two origins on the main chromosome, are differently controlled. A G-rich inverted repeat located in the internal region between the two inverted origin recognition boxes (ORBs) plays as an enhancer for oriC1, whereas the replication initiation at oriC2 is negatively regulated by an ORB-rich region located downstream of oriC2-cdc6E, likely via Cdc6E-titrating. The oriC2 placed on a plasmid is incompatible with the wild-type (but not the ΔoriC2) host strain, further indicating that strict control of the oriC2 activity is important for the cell. This is the first report revealing diverse control mechanisms of origins in haloarchaea, which has provided novel insights into the use and coordination of multiple replication origins in the domain of Archaea.

scholarly journals Analysis of APOBEC-induced mutations in yeast strains with low levels of replicative DNA polymerases

2020 ◽  
Vol 117 (17) ◽  
pp. 9440-9450 ◽  
Author(s):  
Yang Sui ◽  
Lei Qi ◽  
Ke Zhang ◽  
Natalie Saini ◽  
Leszek J. Klimczak ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Induced Mutations ◽  
Single Stranded Dna ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Yeast Strains ◽  
Polymerase Epsilon ◽  
Low Levels ◽  
Lagging Strand

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.

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