scholarly journals CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication.

1997 ◽  
Vol 17 (2) ◽  
pp. 553-563 ◽  
Author(s):  
L Zou ◽  
J Mitchell ◽  
B Stillman
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Genetic Interaction ◽  
Sequence Similarity ◽  
Replication Initiation ◽  
Nonpermissive Temperature ◽  
Functional Connection ◽  
Initiation Of Dna Replication ◽  
Mcm Proteins ◽  
Origin Recognition

The CDC45 gene of Saccharomyces cerevisiae was isolated by complementation of the cold-sensitive cdc45-1 mutant and shown to be essential for cell viability. Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its protein product reveals no significant sequence similarity to any known Mcm family member. Further genetic characterization of the cdc45-1 mutant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. These results not only reveal a functional connection between the origin recognition complex (ORC) and Cdc45p but also extend the CDC45-MCM genetic interaction to all known MCM family members that were shown to be involved in replication initiation. Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry into S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication origins. Furthermore, two-dimensional gels directly showed that chromosomal origins fired less frequently in cdc45-1 cells at the nonpermissive temperature. These findings suggest that Cdc45p, ORC, and Mcm proteins act in concert for replication initiation throughout the genome.

scholarly journals Identification of a Preinitiation Step in DNA Replication That Is Independent of Origin Recognition Complex and cdc6, but Dependent on cdk2

1998 ◽  
Vol 140 (2) ◽  
pp. 271-281 ◽  
Author(s):  
Xuequn Helen Hua ◽  
John Newport
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Minichromosome Maintenance ◽  
Cdk2 Activity ◽  
Proteolytic Pathway ◽  
Egg Extracts ◽  
Dna Replication Origin ◽  
Initiation Of Dna Replication ◽  
Mcm Proteins ◽  
Origin Recognition

Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity.

scholarly journals Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

2001 ◽  
Vol 21 (17) ◽  
pp. 5767-5777 ◽  
Author(s):  
Amit Vas ◽  
Winnie Mok ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Safety Net ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Phosphorylation Sites ◽  
Cdc2 Kinase ◽  
Origin Recognition ◽  
Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

scholarly journals The B-Subunit of DNA Polymerase α-Primase Associates with the Origin Recognition Complex for Initiation of DNA Replication

2004 ◽  
Vol 24 (17) ◽  
pp. 7419-7434 ◽  
Author(s):  
Masashi Uchiyama ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Dna Polymerase ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Dna Polymerase Α ◽  
B Subunit ◽  
Initiation Of Dna Replication ◽  
Primase Complex ◽  
Origin Recognition

ABSTRACT The B-subunit (p70/Pol12p) of the DNA polymerase α-primase (Polα-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polα-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polα-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polα-primase in the initiation of both leading and lagging strands at the replication origins.

scholarly journals Inhibitory effects of acidic phospholipids on the binding of origin-recognition complex to origin DNA

2002 ◽  
Vol 362 (2) ◽  
pp. 395-399 ◽  
Author(s):  
Jong-Ryul LEE ◽  
Masaki MAKISE ◽  
Hitomi TAKENAKA ◽  
Naoko TAKAHASHI ◽  
Yoshihiro YAMAGUCHI ◽  
...  
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Binding Activity ◽  
Atp Binding ◽  
Chromosomal Dna ◽  
Inhibitory Effects ◽  
Nuclear Membranes ◽  
Acidic Phospholipids ◽  
Initiation Of Dna Replication ◽  
Origin Recognition

Origin-recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, shares certain biochemical characteristics with DnaA, the initiator of chromosomal DNA replication in prokaryotes. These similarities include origin-specific DNA binding, ATP binding and ATPase activity. DnaA interacts with acidic phospholipids, such as cardiolipin, and its activity is regulated by these phospholipids. In this study, we examined whether Saccharomyces cerevisiae ORC also interacts with phospholipids. Among the various phospholipids tested, ORC was found to bind specifically to cardiolipin. This binding was inhibited by excess concentrations of salts but unaffected by ATP, adenosine 5′-[γ-thio]triphosphate or the origin DNA. Cardiolipin weakly inhibited the ATP-binding activity of ORC, whereas it strongly inhibited ORC binding to origin DNA. Acidic phospholipids other than cardiolipin (phosphatidylglycerol and phosphatidylinositol) weakly inhibited ORC binding to origin DNA. Furthermore, total phospholipids extracted from yeast nuclear membranes inhibited ORC binding to origin DNA. We consider that phospholipids may modulate initiation of DNA replication in eukaryotes in a similar manner to that found in prokaryotes.

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

1999 ◽  
Vol 112 (12) ◽  
pp. 2011-2018 ◽  
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.

Orc mutants arrest in metaphase with abnormally condensed chromosomes

Development ◽  
2001 ◽  
Vol 128 (9) ◽  
pp. 1697-1707 ◽  
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.

scholarly journals Human Origin Recognition Complex Large Subunit Is Degraded by Ubiquitin-Mediated Proteolysis after Initiation of DNA Replication

2002 ◽  
Vol 9 (3) ◽  
pp. 481-491 ◽  
Author(s):  
Juan Méndez ◽  
X.Helena Zou-Yang ◽  
So-Young Kim ◽  
Masumi Hidaka ◽  
William P. Tansey ◽  
...  
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Large Subunit ◽  
Human Origin ◽  
Initiation Of Dna Replication ◽  
Origin Recognition

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

2019 ◽  
Vol 16 (3) ◽  
pp. 272-277 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Anders Løbner-Olesen
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Cyclic Peptide ◽  
Replication Initiation ◽  
False Positive Result ◽  
Over Expression ◽  
Initiator Protein ◽  
Dna Replication Initiation ◽  
Cellular Processes ◽  
Initiation Of Dna Replication

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

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

2001 ◽  
Vol 21 (8) ◽  
pp. 2790-2801 ◽  
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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