Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability

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.

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Specification of Regions of DNA Replication Initiation during Embryogenesis in the 65-KilobaseDNApolα-dE2F Locus of Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.547 ◽

1999 ◽

Vol 19 (1) ◽

pp. 547-555 ◽

Cited By ~ 88

Author(s):

Takayo Sasaki ◽

Tomoyuki Sawado ◽

Masamitsu Yamaguchi ◽

Tomoyuki Shinomiya

Keyword(s):

Dna Replication ◽

Cultured Cells ◽

Early Stage ◽

Replication Initiation ◽

Replication Origins ◽

Coding Region ◽

Promoter Regions ◽

Dna Replication Initiation ◽

Differentiated Cells ◽

Active Promoter

ABSTRACT In the early stage of Drosophila embryogenesis, DNA replication initiates at unspecified sites in the chromosome. In contrast, DNA replication initiates in specified regions in cultured cells. We investigated when and where the initiation regions are specified during embryogenesis and compared them with those observed in cultured cells by two-dimensional gel methods. In the DNA polymerase α gene (DNApolα) locus, where an initiation region,oriDα, had been identified in cultured Kc cells, repression of origin activity in the coding region was detected after formation of cellular blastoderms, and the range of the initiation region had become confined by 5 h after fertilization. During this work we identified other initiation regions between oriDα and the Drosophila E2F gene (dE2F) downstream of DNApolα. At least four initiation regions showing replication bubbles were identified in the 65-kbDNApolα-dE2F locus in 5-h embryos, but only two were observed in Kc cells. These results suggest that the specification levels of origin usage in 5-h embryos are in the intermediate state compared to those in more differentiated cells. Further, we found a spatial correlation between the active promoter regions fordE2F and the active initiation zones of replication. In 5-h embryos, two known transcripts differing in their first exons were expressed, and two regions close to the respective promoter regions for both transcripts functioned as replication origins. In Kc cells, only one transcript was expressed and functional replication origins were observed only in the region including the promoter region for this transcript.

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DNA Replication Origins Fire Stochastically in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0657 ◽

2006 ◽

Vol 17 (1) ◽

pp. 308-316 ◽

Cited By ~ 133

Author(s):

Prasanta K. Patel ◽

Benoit Arcangioli ◽

Stephen P. Baker ◽

Aaron Bensimon ◽

Nicholas Rhind

Keyword(s):

Dna Replication ◽

Fission Yeast ◽

Replication Initiation ◽

Epigenetic Mechanism ◽

Replication Origins ◽

Origin Firing ◽

Cell Cycles ◽

Initiation Of Replication ◽

Dna Combing ◽

Dna Replication Origins

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

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Investigating the role of Rts1 in DNA replication initiation

Wellcome Open Research ◽

10.12688/wellcomeopenres.13884.1 ◽

2018 ◽

Vol 3 ◽

pp. 23 ◽

Cited By ~ 1

Author(s):

Ana B.A. Wallis ◽

Conrad A. Nieduszynski

Keyword(s):

Dna Replication ◽

Temperature Sensitivity ◽

Protein Phosphatase ◽

Dna Helicase ◽

Replication Initiation ◽

Replication Origins ◽

Origin Firing ◽

Replication Factor ◽

Dna Replication Initiation ◽

Genomic Screen

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2. Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains. Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

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

10.1101/174730 ◽

2017 ◽

Cited By ~ 1

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.

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Isolation of Broad-Host-Range Replicons from Marine Sediment Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.64.8.2822-2830.1998 ◽

1998 ◽

Vol 64 (8) ◽

pp. 2822-2830 ◽

Cited By ~ 53

Author(s):

Patricia A. Sobecky ◽

Tracy J. Mincer ◽

Michelle C. Chang ◽

Aresa Toukdarian ◽

Donald R. Helinski

Keyword(s):

Sequence Analysis ◽

Marine Sediments ◽

Marine Sediment ◽

Replication Initiation ◽

Plasmid Replication ◽

Broad Host Range ◽

Bacterial Isolates ◽

Replication Origins ◽

Naturally Occurring ◽

Sediment Bacteria

ABSTRACT Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888–895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the α and γ subclasses of the classProteobacteria. Three of the marine sediment isolates are related to the γ-3 subclass organisms Vibrio splendidusand Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication inEscherichia coli DH5α and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the generaAchromobacter, Pseudomonas,Serratia, and Vibrio. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.

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Linear Derivatives of Saccharomyces cerevisiae Chromosome III Can Be Maintained in the Absence of Autonomously Replicating Sequence Elements

Molecular and Cellular Biology ◽

10.1128/mcb.01246-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4652-4663 ◽

Cited By ~ 31

Author(s):

Ann Dershowitz ◽

Marylynn Snyder ◽

Mohammed Sbia ◽

Joan H. Skurnick ◽

Loke Y. Ong ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Loss Rate ◽

Replication Initiation ◽

Replication Origins ◽

Additional Increase ◽

Autonomously Replicating Sequence ◽

Sequence Elements ◽

Chromosome Fragments ◽

Chromosome Iii ◽

The Right

ABSTRACT Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.

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Multiple Replication Origins of Halobacterium sp. Strain NRC-1: Properties of the Conserved orc7-Dependent oriC1

Journal of Bacteriology ◽

10.1128/jb.00210-09 ◽

2009 ◽

Vol 191 (16) ◽

pp. 5253-5261 ◽

Cited By ~ 21

Author(s):

James A. Coker ◽

Priya DasSarma ◽

Melinda Capes ◽

Tammitia Wallace ◽

Karen McGarrity ◽

...

Keyword(s):

Binding Sites ◽

Replication Initiation ◽

Replication Origins ◽

Circular Chromosome ◽

Autonomously Replicating Sequence ◽

Initiation Point ◽

Point Analysis ◽

Gene Orthologs ◽

Marker Frequency ◽

Multiple Replication

ABSTRACT The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.

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