scholarly journals The dynamics of replication licensing in live Caenorhabditis elegans embryos

2012 ◽  
Vol 196 (2) ◽  
pp. 233-246 ◽  
Author(s):  
Remi Sonneville ◽  
Matthieu Querenet ◽  
Ashley Craig ◽  
Anton Gartner ◽  
J. Julian Blow
Keyword(s):  
Caenorhabditis Elegans ◽  
Dna Replication ◽  
Dna Binding ◽  
Feedback Loop ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Chromosomal Dna ◽  
M Phase ◽  
Prereplicative Complex

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2–7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2–7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP–ORC-1 bound chromatin independently of ORC-2–5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2–7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2–7 complexes. MCM-2–7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2–7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.

scholarly journals Cyclin B-Cdk1 Kinase Stimulates ORC- and Cdc6-Independent Steps of Semiconservative Plasmid Replication in Yeast Nuclear Extracts

1999 ◽  
Vol 19 (2) ◽  
pp. 1226-1241 ◽  
Author(s):  
Bernard P. Duncker ◽  
Philippe Pasero ◽  
Diego Braguglia ◽  
Patrick Heun ◽  
Michael Weinreich ◽  
...  
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Autonomously Replicating Sequence ◽  
Nuclear Extracts ◽  
M Phase ◽  
Efficient Replication ◽  
Prereplicative Complex

ABSTRACT Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40 SIC1 , very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40 SIC1 , restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

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

2012 ◽  
Vol 287 (15) ◽  
pp. 11891-11898 ◽  
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.

scholarly journals Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

1998 ◽  
Vol 9 (1) ◽  
pp. 63-73 ◽  
Author(s):  
Antonia Lopez-Girona ◽  
Odile Mondesert ◽  
Janet Leatherwood ◽  
Paul Russell
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Fission Yeast ◽  
Phosphorylation Site ◽  
Constitutive Expression ◽  
S Phase ◽  
Negative Regulation ◽  
Chromosomal Dna

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

scholarly journals Xenopus Cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading

2000 ◽  
Vol 14 (12) ◽  
pp. 1528-1540
Author(s):  
Pedro Jares ◽  
J. Julian Blow
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Protein Complexes ◽  
Present Report ◽  
S Phase ◽  
Chromosomal Dna ◽  
Minichromosome Maintenance ◽  
Initiation Of Replication ◽  
Sequential Binding

The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication. The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin. Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase. In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs. We show that XCdc7 binds to chromatin during G1 and S phase. XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing. Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete. Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function. From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system.

Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle

1993 ◽  
Vol 106 (3) ◽  
pp. 983-994 ◽  
Author(s):  
F. Fang ◽  
J.W. Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Protein ◽  
Model System ◽  
S Phase ◽  
Chromosomal Dna ◽  
Cdc2 Kinase ◽  
Single Stranded Dna

RP-A is a single-stranded DNA-binding protein, which has been shown to be required for DNA replication using an SV40 model system. The protein has also been shown to be phosphorylated at the G1-S phase transition. Using Xenopus cell-free extracts we have investigated the role of RP-A in nuclear replication and characterized the kinases and conditions that lead to phosphorylation of RP-A during the cell cycle. By immunodepleting RP-A from Xenopus extracts we have shown that RP-A is essential for replication of chromosomal DNA. Our results show that, during S phase, only that RP-A which is associated with nuclei is phosphorylated. Furthermore our results indicate that during S phase RP-A is only phosphorylated when associated with single-stranded DNA. By immunodepleting cdk2 kinase we show that cdk2 kinase is required for the observed phosphorylation of RP-A in nuclei during S phase. However, using purified cdk2 kinase and RP-A we are unable to detect a direct phosphorylation of RP-A by cdk2 kinase. This observation suggests that phosphorylation of DNA-bound RP-A at S phase is carried out by a kinase distinct from cdk2. Consistent with this we find that when single-stranded DNA is added to S phase extracts depleted of cdk2 kinase, RP-A is phosphorylated. Together these results suggest that cdk2 kinase participates in the activation of DNA replication at a stage prior to the binding of RP-A to the initiation complex. In addition to RP-A phosphorylation in S phase, we have also found that at the onset of mitosis RP-A is quantitatively phosphorylated and that phosphorylation is directly mediated by cdc2 kinase. However, at this time during the cell cycle, cdc2-dependent phosphorylation of RP-A is independent of DNA binding. These observations further demonstrate the distinctions between cdk2 and cdc2 kinases.

Analysis of mutant origin recognition complex with reduced ATPase activity in vivo and in vitro

2008 ◽  
Vol 413 (3) ◽  
pp. 535-543 ◽  
Author(s):  
Masaya Takehara ◽  
Masaki Makise ◽  
Hitomi Takenaka ◽  
Teita Asano ◽  
Tohru Mizushima
Keyword(s):  
Atpase Activity ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
Aaa Atpases ◽  
Origin Recognition

In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA+ (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Orc1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA+ proteins. Plasmid-shuffling analysis revealed that Asn600, Arg694 and Arg704 are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg694→Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg694 of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.

scholarly journals Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

2007 ◽  
Vol 27 (8) ◽  
pp. 3143-3153 ◽  
Author(s):  
Maxim Balasov ◽  
Richard P. H. Huijbregts ◽  
Igor Chesnokov
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Dominant Negative ◽  
The Core ◽  
Culture Cells ◽  
Replication Domain ◽  
Origin Recognition

ABSTRACT The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

scholarly journals Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro

2000 ◽  
Vol 14 (13) ◽  
pp. 1631-1641 ◽  
Author(s):  
Tohru Mizushima ◽  
Naoko Takahashi ◽  
Bruce Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Binding Activity ◽  
Chromosomal Dna ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Dna Replication Origins ◽  
Origin Recognition

An interaction between the origin recognition complex (ORC) and Cdc6p is the first and a key step in the initiation of chromosomal DNA replication. We describe the assembly of an origin-dependent complex containing ORC and Cdc6p from Saccharomyces cerevisiae. Cdc6p increases the DNA binding specificity of ORC by inhibiting non-specific DNA binding of ORC. Cdc6p induces a concomitant change in the conformation of ORC and mutations in the Cdc6p Walker A and Walker B motifs, or ATP-γ-S inhibited these activities of Cdc6p. These data suggest that Cdc6p modifies ORC function at DNA replication origins. On the basis of these results in yeast, we propose that Cdc6p may be an essential determinant of origin specificity in metazoan species.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

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