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

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.

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Genome-Wide Identification of Early-Firing Human Replication Origins by Optical Replication Mapping

10.1101/214841 ◽

2017 ◽

Cited By ~ 11

Author(s):

Kyle Klein ◽

Weitao Wang ◽

Tyler Borrman ◽

Saki Chan ◽

Denghong Zhang ◽

...

Keyword(s):

Single Molecule ◽

Replication Timing ◽

S Phase ◽

Replication Initiation ◽

Replication Origins ◽

Origin Firing ◽

Efficient Replication ◽

Mapping Technology ◽

Low Efficiency ◽

Early Replication

AbstractThe timing of DNA replication is largely regulated by the location and timing of replication origin firing. Therefore, much effort has been invested in identifying and analyzing human replication origins. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual origins in metazoans has made mapping the location and timing of replication initiation in human cells difficult. We have mapped early-firing origins in HeLa cells using Optical Replication Mapping, a high-throughput single-molecule approach based on Bionano Genomics genomic mapping technology. The single-molecule nature and 290-fold coverage of our dataset allowed us to identify origins that fire with as little as 1% efficiency. We find sites of human replication initiation in early S phase are not confined to well-defined efficient replication origins, but are instead distributed across broad initiation zones consisting of many inefficient origins. These early-firing initiation zones co-localize with initiation zones inferred from Okazaki-fragment-mapping analysis and are enriched in ORC1 binding sites. Although most early-firing origins fire in early-replication regions of the genome, a significant number fire in late-replicating regions, suggesting that the major difference between origins in early and late replicating regions is their probability of firing in early S-phase, as opposed to qualitative differences in their firing-time distributions. This observation is consistent with stochastic models of origin timing regulation, which explain the regulation of replication timing in yeast.

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Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

Journal of Biological Chemistry ◽

10.1074/jbc.m111.338467 ◽

2012 ◽

Vol 287 (15) ◽

pp. 11891-11898 ◽

Cited By ~ 27

Author(s):

Kyung Yong Lee ◽

Sung Woong Bang ◽

Sang Wook Yoon ◽

Seung-Hoon Lee ◽

Jong-Bok Yoon ◽

...

Keyword(s):

Cell Cycle ◽

Origin Recognition Complex ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Replication Origins ◽

Eukaryotic Chromosome ◽

M Phase ◽

Cycle Dependent ◽

Prereplicative Complex ◽

Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

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Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins

Journal of Cell Science ◽

10.1242/jcs.112.12.2011 ◽

1999 ◽

Vol 112 (12) ◽

pp. 2011-2018 ◽

Cited By ~ 2

Author(s):

A. Rowles ◽

S. Tada ◽

J.J. Blow

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Replication Origin ◽

Origin Recognition Complex ◽

S Phase ◽

Replication Origins ◽

Free System ◽

Essential Function ◽

Initiation Of Dna Replication ◽

Origin Recognition

During late mitosis and early G1, a series of proteins are assembled onto replication origins that results in them becoming ‘licensed’ for replication in the subsequent S phase. In Xenopus this first involves the assembly onto chromatin of the Xenopus origin recognition complex XORC, and then XCdc6, and finally the RLF-M component of the replication licensing system. In this paper we examine changes in the way that XORC associates with chromatin in the Xenopus cell-free system as origins become licensed. Restricting the quantity of XORC on chromatin reduced the extent of replication as expected if a single molecule of XORC is sufficient to specify a single replication origin. During metaphase, XOrc1 associated only weakly with chromatin. In early interphase, XOrc1 formed a strong complex with chromatin, as evidenced by its resistance to elution by 200 mM salt, and this state persisted when XCdc6 was assembled onto the chromatin. As a consequence of origins becoming licensed the association of XOrc1 and XCdc6 with chromatin was destabilised, and XOrc1 became susceptible to removal from chromatin by exposure to either high salt or high Cdk levels. At this stage the essential function for XORC and XCdc6 in DNA replication had already been fulfilled. Since high Cdk levels are required for the initiation of DNA replication, this ‘licensing-dependent origin inactivation’ may contribute to mechanisms that prevent re-licensing of replication origins once S phase has started.

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Conversion of a Replication Origin to a Silencer through a Pathway Shared by a Forkhead Transcription Factor and an S Phase Cyclin

Molecular Biology of the Cell ◽

10.1091/mbc.e07-04-0323 ◽

2008 ◽

Vol 19 (2) ◽

pp. 608-622 ◽

Cited By ~ 14

Author(s):

Laurieann Casey ◽

Erin E. Patterson ◽

Ulrika Müller ◽

Catherine A. Fox

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Origin Recognition Complex ◽

S Phase ◽

Forkhead Transcription Factor ◽

Origin Firing ◽

Type Locus ◽

Sir Proteins ◽

Dna Elements ◽

A Cell

Silencing of the mating-type locus HMR in Saccharomyces cerevisiae requires DNA elements called silencers. To establish HMR silencing, the origin recognition complex binds the HMR-E silencer and recruits the silent information regulator (Sir)1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2–4. However, silencing is semistable even in sir1Δ cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1hc), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5Δ). FKH1hc caused only a modest increase in Fkh1 levels but effectively reestablished Sir2–4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1hc prolonged the cell cycle in a manner distinct from deletion of its close paralogue FKH2, and it created a cell cycle phenotype more reminiscent to that caused by a clb5Δ. Unexpectedly, and in contrast to SIR1, both FKH1hc and clb5Δ established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRΔE::ARS). HMRΔE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRΔE::ARS1 was reduced by clb5Δ or FKH1hc, whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRΔE::ARS1 did not result from formation of Sir2–4 chromatin because sir2Δ did not rescue origin firing in clb5Δ cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2–4 chromatin and late-origin firing through opposing regulation of a common pathway.

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Orc mutants arrest in metaphase with abnormally condensed chromosomes

Development ◽

10.1242/dev.128.9.1697 ◽

2001 ◽

Vol 128 (9) ◽

pp. 1697-1707 ◽

Cited By ~ 5

Author(s):

M.F. Pflumm ◽

M.R. Botchan

Keyword(s):

Dna Replication ◽

Origin Recognition Complex ◽

Metaphase Plate ◽

Mitotic Checkpoint ◽

Early Embryo ◽

S Phase ◽

Replication Initiation ◽

General Role ◽

Chromosomal Condensation ◽

Origin Recognition

The origin recognition complex (ORC) is a six subunit complex required for eukaryotic DNA replication initiation and for silencing of the heterochromatic mating type loci in Saccharomyces cerevisiae. Our discovery of the Drosophila ORC complex concentrated in the centric heterochromatin of mitotic cells in the early embryo and its interactions with heterochromatin protein 1 (HP-1) lead us to speculate that ORC may play some general role in chromosomal folding. To explore the role of ORC in chromosomal condensation, we have identified a mutant of subunit 5 of the Drosophila melanogaster origin recognition complex (Orc5) and have characterized the phenotypes of both the Orc5 and the previously identified Orc2 mutant, k43. Both Orc mutants died at late larval stages and surprisingly, despite a reduced number of S-phase cells, an increased fraction of cells were also detected in mitosis. For this latter population of cells, Orc mutants arrest in a defective metaphase with shorter and thicker chromosomes that fail to align at the metaphase plate within a poorly assembled mitotic spindle. In addition, sister chromatid cohesion was frequently lost. PCNA and MCM4 mutants had similar phenotypes to Orc mutants. We propose that DNA replication defects trigger the mitotic arrest, due to the fact that frequent fragmentation was observed. Thus, cells have a mitotic checkpoint that senses chromosome integrity. These studies also suggest that the density of functional replication origins and completion of S phase are requirements for proper chromosomal condensation.

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Influence of Origin Recognition Complex Proteins on the Copy Numbers of Three Chromosomes in Haloferax volcanii

Journal of Bacteriology ◽

10.1128/jb.00161-18 ◽

2018 ◽

Vol 200 (17) ◽

Cited By ~ 4

Author(s):

Katharina Ludt ◽

Jörg Soppa

Keyword(s):

Copy Number ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Haloferax Volcanii ◽

Deletion Mutants ◽

Replication Origins ◽

Content Type ◽

Chromosome Copy Number ◽

Copy Numbers ◽

Origin Recognition

ABSTRACT Replication initiation in archaea involves a protein named ORC, Cdc6, or ORC1/Cdc6, which is homologous to the eukaryotic origin recognition complex (ORC) proteins and to the eukaryotic Cdc6. Archaeal replication origins are comprised of origin repeat regions and adjacent orc genes. Some archaea contain a single replication origin and a single orc gene, while others have more than one of each. Haloferax volcanii is exceptional because it contains, in total, six replication origins on three chromosomes and 16 orc genes. Phylogenetic trees were constructed that showed that orc gene duplications occurred at very different times in evolution. To unravel the influence of the ORC proteins on chromosome copy number and cellular fitness, it was attempted to generate deletion mutants of all 16 genes. A total of 12 single-gene deletion mutants could be generated, and only three orc gene turned out to be essential. For one gene, the deletion analysis failed. Growth analyses revealed that no deletion mutant had a growth defect, but some had a slight growth advantage compared to the wild type. Quantification of the chromosome copy numbers in the deletion mutants showed that all 12 ORC proteins influenced the copy numbers of one, two, or all three chromosomes. The lack of an ORC led to an increase or decrease of chromosome copy number. Therefore, chromosome copy numbers in Hfx. volcanii are regulated by an intricate network of ORC proteins. This is in contrast to other archaea, in which ORC proteins typically bind specifically to the adjacent origin. IMPORTANCE The core origins of archaea are comprised of a repeat region and an adjacent gene for an origin recognition complex (ORC) protein, which is homologous to eukaryotic ORC proteins. Haloferax volcanii is exceptional because it contains six replication origins on three chromosomes and an additional 10 orc genes that are not adjacent to an origin. This unique ORC protein repertoire was used to unravel the importance of core origin orc genes and of origin-remote orc genes. Remarkably, all ORC proteins influenced the copy number of at least one chromosome. Some of them influenced those of all three chromosomes, showing that cross-regulation in trans exists in Hfx. volcanii. Furthermore, the evolution of the archaeal ORC protein family was analyzed.

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Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2790-2801.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2790-2801 ◽

Cited By ~ 31

Author(s):

James F. Theis ◽

Carol S. Newlon

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Binding Site ◽

Binding Sites ◽

Origin Recognition Complex ◽

Replication Origins ◽

Discrete Site ◽

Origin Function ◽

Single Binding Site ◽

Origin Recognition

ABSTRACT While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins inSaccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

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Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions

International Journal of Molecular Sciences ◽

10.3390/ijms22073481 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3481

Author(s):

Afaf Eladl ◽

Yudai Yamaoki ◽

Shoko Hoshina ◽

Haruka Horinouchi ◽

Keiko Kondo ◽

...

Keyword(s):

Fluorescence Anisotropy ◽

Binding Sites ◽

Replication Origin ◽

Origin Recognition Complex ◽

Chemical Shift Perturbation ◽

Human Origin ◽

Replication Origins ◽

Genome Wide ◽

G Quadruplex ◽

Origin Recognition

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413–511 of human ORC subunit 1, hORC1413–511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413–511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413–511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413–511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413–511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

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Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms

10.1101/2020.07.04.187286 ◽

2020 ◽

Cited By ~ 3

Author(s):

Y. Hu ◽

A. Tareen ◽

Y-J. Sheu ◽

W. T. Ireland ◽

C. Speck ◽

...

Keyword(s):

Dna Replication ◽

Gene Silencing ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Separate Analysis ◽

Genome Replication ◽

Sequence Specificity ◽

Sequence Motifs ◽

Origin Firing ◽

Α Helix

AbstractDNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

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High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

10.1101/2021.05.14.443897 ◽

2021 ◽

Author(s):

Dashiell J Massey ◽

Amnon Koren

Keyword(s):

Dna Replication ◽

Replication Origin ◽

Replication Timing ◽

S Phase ◽

Human Cells ◽

Replication Initiation ◽

High Throughput Analysis ◽

Timing Variability ◽

Fixed Set ◽

Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

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