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PubMed1997

The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence.

Autonomously replicating sequence (ARS) elements, which function as the cis-acting chromosomal replicators in the yeast Saccharomyces cerevisiae, depend upon an essential copy of the 11-bp ARS consensus sequence (ACS) for activity. Analysis of the chromosome III replicator ARS309 unexpectedly revealed that its essential ACS differs from the canonical ACS at two positions. One of the changes observed in ARS309 inactivates other ARS elements. This atypical ACS binds the origin recognition complex efficiently and is required for chromosomal replication origin activity. Comparison of the essential ACS of ARS309 with the essential regions of other ARS elements revealed an expanded 17-bp conserved sequence that efficiently predicts the essential core of ARS elements.

Base Sequence↗
PubMed2004

A Xenopus Dbf4 homolog is required for Cdc7 chromatin binding and DNA replication.

BACKGROUND: Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase. RESULTS: We have cloned a Xenopus homologue of Dbf4 (XDbf4), the sequence of which confirms the results of Furukhori et al. We have analysed the role of XDbf4 in DNA replication using cell-free extracts of Xenopus eggs. Our results indicate that XDbf4 is the regulatory subunit of XCdc7 required for DNA replication. We show that XDbf4 binds to chromatin during interphase, but unlike XCdc7, its chromatin association is independent of pre-RC formation, occurring in the absence of licensing, XCdc6 and XORC. Moreover, we show that the binding of XCdc7 to chromatin is dependent on the presence of XDbf4, whilst under certain circumstances XDbf4 can bind to chromatin in the absence of XCdc7. We provide evidence that the chromatin binding of XDbf4 that occurs in the absence of licensing depends on checkpoint activation. CONCLUSIONS: We have identified XDbf4 as a functional activator of XCdc7, and show that it is required to recruit XCdc7 to chromatin. Our results also suggest that XCdc7 and XDbf4 are differentially regulated, potentially responding to different cell cycle signals.

Amino Acid Sequence↗
PubMed2003

The Drosophila HOAP protein is required for telomere capping.

HOAP (HP1/ORC-associated protein) has recently been isolated from Drosophila melanogaster embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). Here, we show that caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. Our analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2.

Animals↗
PubMed2006

A Dbf4p BRCA1 C-terminal-like domain required for the response to replication fork arrest in budding yeast.

Dbf4p is an essential regulatory subunit of the Cdc7p kinase required for the initiation of DNA replication. Cdc7p and Dbf4p orthologs have also been shown to function in the response to DNA damage. A previous Dbf4p multiple sequence alignment identified a conserved approximately 40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called "motif N." BRCT motifs encode approximately 100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved approximately 100-residue N-terminal region of Dbf4p that includes motif N but is capable of encoding a single BRCT-like domain. Dbf4p orthologs diverge from the BRCT motif at the C terminus but may encode a similar secondary structure in this region. We have therefore called this the BRCT and DBF4 similarity (BRDF) motif. The principal role of this Dbf4p motif was in the response to replication fork (RF) arrest; however, it was not required for cell cycle progression, activation of Cdc7p kinase activity, or interaction with the origin recognition complex (ORC) postulated to recruit Cdc7p-Dbf4p to origins. Rad53p likely directly phosphorylated Dbf4p in response to RF arrest and Dbf4p was required for Rad53p abundance. Rad53p and Dbf4p therefore cooperated to coordinate a robust cellular response to RF arrest.

Amino Acid Motifs↗
PubMed1996

Replication origins in eukaroytes.

Recent experiments in budding yeast and Xenopus have provided new insights into the regulation of eukaroytic DNA replication. The multi-subunit origin recognition complex plays a key role in initiation, remaining bound at origins of replication during most of the cell cycle. Early in the cell cycle, Cdc6 and the Mcm proteins 'reset' chromatin for another round of DNA replication. Cyclin-dependent kinases appear to play a dual role, both in activating replication origins and blocking the formation of new pre-replicative complexes; thus limiting replication to once per cell cycle.

Animals↗
PubMed1994

Replication and transcription. Silence of the ORCs.

The origin recognition complex, a multi-protein complex known to bind to replication origins, has now been implicated in transcriptional silencing, providing another link between DNA replication and transcription.

Base Sequence↗
PubMed2005

Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.

Antineoplastic Agents, Alkylating↗
PubMed2001

Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins.

BACKGROUND: In order to ensure precise chromosome duplication, eukaryotes "license" their replication origins during late mitosis and early G1 by assembling complexes of Mcm2-7 onto them. Mcm2-7 are essential for DNA replication, but are displaced from origins as they initiate, thus ensuring that no origin fires more than once in a single cell cycle. RESULTS: Here we show that a combination of purified nucleoplasmin, the origin recognition complex (ORC), Cdc6, RLF-B/Cdt1 and Mcm2-7 can promote functional origin licensing and the assembly of Mcm2-7 onto Xenopus sperm nuclei. The reconstituted reaction is inhibited by geminin, a specific RLF-B/Cdt1 inhibitor. Interestingly, the purified ORC used in the reconstitution had apparently lost the Orc6 subunit, suggesting that Orc6 is not essential for replication licensing. We use the reconstituted system to make a preliminary analysis of the different events occurring during origin assembly, and examine their nucleotide requirements. We show that the loading of Xenopus ORC onto chromatin is strongly stimulated by both ADP, ATP and ATP-gamma-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-gamma-S. CONCLUSIONS: Nucleoplasmin, ORC, Cdc6, RLF-B/Cdt1 and Mcm2-7 are the only proteins required for functional licensing and the loading of Mcm2-7 onto chromatin. The requirement for nucleoplasmin probably only reflects a requirement to decondense sperm chromatin before ORC can bind to it. Use of this reconstituted system should allow a full biochemical analysis of origin licensing and Mcm2-7 loading.

Animals↗
PubMed1997

Getting started: regulating the initiation of DNA replication in yeast.

Initiation of DNA replication in yeast appears to operate through a two-step process. The first step occurs at the end of mitosis in the previous cell cycle, where, following the decrease in B cyclin-dependent kinase activity, an extended protein complex called the prereplicative complex (pre-RC) forms over the origin of replication. This complex is dependent on the association of the Cdc6 protein with the Origin Recognition Complex (ORC) and appears concomitantly with the nuclear entry of members of the Mcm family of proteins. The second step is dependent upon the cell passing through a G1 decision point called Start. If the environmental conditions are favorable, and the cells reach a critical size, then there is a rise in G1 cyclin-dependent kinase activity, which leads to the activation of downstream protein kinases; the protein kinases are, in turn, required for triggering initiation from the preformed initiation complexes. These protein kinases, Dbf4-Cdc7 and Clb5/6(B-cyclin)-Cdc28, are thought to phosphorylate targets within the pre-RC. The subsequent rise in B cyclin protein kinase activity following Start not only triggers origin firing, but also inhibits the formation of new pre-RCs, which ensures that there is only one S phase in each cell cycle. The destruction of B-cyclin protein kinase activity at the end of the cell cycle potentiates the formation of new pre-RCs-resetting origins for the next S phase.

Carrier Proteins↗
PubMed2002

The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication.

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat-containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.

Animals↗
PubMed2000

Think global, act local--how to regulate S phase from individual replication origins.

All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved. Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins. Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases. The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins. Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states.

Animals↗
PubMed2001

Nucleosomes positioned by ORC facilitate the initiation of DNA replication.

The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events. To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S. cerevisiae replication origins. Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function.

Chromatin↗
PubMed1992

Protein-DNA interactions at a yeast replication origin.

An understanding of the protein-DNA interactions in vivo at origins of DNA replication in eukaryotes is essential to delineate the mechanism of initiation of DNA synthesis and its control in the cell cycle. In the yeast Saccharomyces cerevisiae, a family of sequences known as autonomously replicating sequences (ARSs) function as origins of bidirectional DNA replication on plasmids and, in several instances, also in their normal chromosomal location. Here we use nucleotide resolution genomic footprinting to investigate the association of proteins with ARS1. Nuclease protection patterns indicate that at least two different cellular factors interact with functional elements in ARS1. The first seems to be ARS-binding factor 1. The second seems to be a novel protein that generates extensive protection over the essential ARS consensus sequence and phased DNaseI-sensitive sites across a functionally important flanking sequence. Hypersensitivity of this region to cleavage by copper phenanthroline indicates that it is under torsional strain, analogous to that produced at transcriptional start sites by assembly of an initiation complex. The protection in situ is similar to that generated by the origin recognition complex (ORC) protein.

Base Sequence↗
PubMed2002

In the beginning: a viral origin exploits the cell.

The origin of replication of Epstein-Barr Virus (EBV) has been shown to associate with cellular proteins that regulate the initiation of DNA synthesis in human cells. These cellular proteins are subunits of both the origin recognition complex and what might be the replicative helicase. Such associations identify the EBV origin of replication as being an experimentally tractable paradigm for mammalian replicons.

DNA Helicases↗
PubMed2006

Regulation of replication licensing by acetyltransferase Hbo1.

The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.

Acetylation↗
PubMed1992

DNA replication and the cell cycle.

The replication of DNA in the eukaryotic cell cycle is one of the most highly regulated events in cell growth and division. Biochemical studies on the replication of the genome of the small DNA virus simian virus 40 (SV40) have resulted in the identification of a number of DNA replication proteins from human cells. One of these, Replication Protein A (RPA), was phosphorylated in a cell cycle-dependent manner, beginning at the onset of DNA replication. RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase. This kinase also stimulated the unwinding of the SV40 origin of DNA replication during initiation of DNA replication in vitro, suggesting a mechanism by which cdc2 kinase may regulate DNA replication. Functional homologues of the DNA replication factors have been identified in extracts from the yeast Saccharomyces cerevisiae, enabling a genetic characterization of the role of these proteins in the replication of cellular DNA. A cellular origin binding protein had not been characterized. To identify proteins that function like T antigen at cellular origins of DNA replication, we examined the structure of a yeast origin of DNA replication in detail. This origin consists of four separate functional elements, one of which is essential. A multiprotein complex that binds to the essential element has been identified and purified. This protein complex binds to all known cellular origins from S. cerevisiae and may function as an origin recognition complex.

Animals↗
PubMed1999

The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin.

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.

Animals↗
PubMed2002

Identification of Tah11/Sid2 as the ortholog of the replication licensing factor Cdt1 in Saccharomyces cerevisiae.

Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin licensing. Here we identify Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. We show that sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. We conclude that origin licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Amino Acid Sequence↗