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PubMed2001

The Sir1 protein's association with a silenced chromosome domain.

Silencing of the cryptic mating-type locus HMR requires recognition of a small DNA sequence element, the HMR-E silencer, by the Sir1p, one of four Sir proteins required for the assembly of silenced chromatin domains in Saccharomyces cerevisiae. The Sir1p recognizes the silencer through interactions with the origin recognition complex (ORC), a protein complex that binds the silencer DNA directly. Sir1p was physically associated with HMR in chromatin, and this association required a Sir1p-ORC interaction, suggesting that it reflected the Sir1p silencer-recognition function required for silencing. Sir1p was not associated with nonsilencer replication origins that bind the ORC, indicating that a Sir1p-ORC interaction is confined to silencers. Significantly, the other SIR genes were required for Sir1p's association with HMR. Thus, multiple protein contacts required for and unique to silent chromatin may confine a Sir1p-ORC interaction to silencers. The Sir1p was present at extremely low concentrations in yeast cells yet was associated with HMR at all stages of the cell cycle examined. These data provide insights into the mechanisms that establish and restrict the assembly of silenced chromatin to only a few discrete chromosomal domains.

Base Sequence↗
PubMed1998

Discrete start sites for DNA synthesis in the yeast ARS1 origin.

Sites of DNA synthesis initiation have been detected at the nucleotide level in a yeast origin of bidirectional replication with the use of replication initiation point mapping. The ARS1 origin of Saccharomyces cerevisiae showed a transition from discontinuous to continuous DNA synthesis in an 18-base pair region (nucleotides 828 to 845) from within element B1 toward B2, adjacent to the binding site for the origin recognition complex, the putative initiator protein.

Base Composition↗
PubMed1999

AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes.

Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously. Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, the Halobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase. Alignment of these sequences, in light of the structures of the clamp loader delta' subunit of Escherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class. Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life. These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA-protein complexes.

Adenosine Triphosphatases↗
PubMed1998

[Is the replicon model applicable to higher eukaryotes?].

Thirty-five years ago, the Replicon model was proposed by Jacob, Brenner and Cuzin to explain the regulation of the Escherichia coli DNA replication. In this model, a genetic element, the replicator, would function as a target for a positive-acting initiator protein to drive the initiation of replication. This simple idea has been extremely useful in providing a framework to explain how the initiation of DNA replication occurs in all organisms. The identification of autonomously replicating sequences (ARSs) in budding yeast was the first extension of the Replicon model to eukaryotic chromosomes. In the higher eukaryotes, many biochemically defined replication start sites have been identified; nevertheless there is little genetic data indicating that these sites contain DNA sequences that are essential for replication. Moreover, in early Xenopus or Drosophila embryos, specific DNA sequences are not required either for initiating DNA replication or for preventing rereplication within a single cell cycle. This apparently fundamental difference between replicators in yeast and metazoan embryos may be more superficial than initially thought. In fact, during the past several years, an eukaryotic initiator conserved from yeast to man and also present in embryonic cells, the origin recognition complex (ORC), has been characterized, suggesting that the initiation mechanism should be essentially the same in prokaryotes and eukaryotes. In addition, the efficient once-per-cell-cycle replication of DNA is ensured in eukaryotes by a simple two-step mechanism in which the assembly of stable prereplicative complexes (PreRCs) at origins precedes and is temporally separated from the firing of these origins. Regulation of this process by cyclin-dependent kinases ensures that when origins fire, the cell is no longer competent to form new PreRCs. Now, it is important to understand how these complexes are remodeled or disassembled during replication initiation to trigger the transition from a stable origin-bound complex to a mobile replication machine.

Animals↗
PubMed2003

Genome-wide distribution of DNA replication origins at A+T-rich islands in Schizosaccharomyces pombe.

Genome-wide analysis of replication dynamics requires the previous identification of DNA replication origins (ORIs). However, variability among the ORIs makes it difficult to predict their distribution across the genome on the basis of their sequence. We report here that ORIs in Schizosaccharomyces pombe coincide with discrete chromosomal A+T-rich islands of up to 1 kb long that are characterized by a distinctive A+T content that clearly differentiates them from the rest of the genome. Genome-wide analysis has enabled us to identify 384 of these regions, which predicts the position of most ORIs in the genome, as shown by functional replication analyses. A+T-rich islands occur at the mating locus, centromeres and subtelomeric regions at a density that is approximately fourfold higher than elsewhere in the genome, which suggests a link between the origin recognition complex (ORC) and transcriptional silencing in these regions. The absence of consensus elements in A+T-rich islands implies that different sequences can target the ORC to different ORIs.

AT Rich Sequence↗
PubMed2002

A novel chk1-dependent G1/M checkpoint in fission yeast.

Fission yeast cells with a temperature-sensitive Orp1 protein, a component of the origin recognition complex, cannot perform DNA replication at the restrictive temperature. Seventy percent of orp1-4 cells arrest with a 1C DNA content, whereas 30% proceed to mitosis ('cut'). The arrest depends upon the checkpoint Rad proteins and, surprisingly, the Chk1 protein, which is thought to act only from late S phase. The arrested cells maintain a 1C DNA content, as judged by flow cytometry, and the early origin ars3001 has not been initiated, as judged by 2D gel analysis. We show that in G1-arrested orp1-4 cells, Wee1 phosphorylates and inactivates Cdc2. Activation of Chk1 occurs earlier than Cdc2 phosphorylation, indicating a novel role for Chk1, namely to induce and/or maintain Cdc2 phosphorylation upon checkpoint activation in G1. We also show that commitment to cutting occurs already in early G1 phase.

CDC2 Protein Kinase↗
PubMed2005

Eukaryotic origins of DNA replication: could you please be more specific?

Initiation of eukaryotic DNA replication commences when the origin recognition complex (ORC) binds to DNA, recruiting helicases, polymerases, and necessary cofactors. While the biochemical mechanism and factors involved in replication initiation appear to be highly conserved, the DNA sequences at which these events take place in different organisms are not. Thus, while ORC appears to bind to specific DNA sequences in budding yeast, there is increasing new evidence that metazoan ORC complexes do not rely on sequence to be directed to origins of replication. Here, we review examples of specific and non-specific initiation, and we consider what, if not DNA sequence, accounts for DNA binding of ORC to defined regions in eukaryotic genomes.

Acetylation↗
PubMed2002

Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity.

DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.

Amino Acid Sequence↗
PubMed1996

Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome.

A yeast autonomously replicating sequence, ARS305, shares essential components with a chromosome III replicator, ORI305. Known components include an ARS consensus sequence (ACS) element, presumed to bind the origin recognition complex (ORC), and a broad 3'-flanking sequence which contains a DNA unwinding element. Here linker substitution mutagenesis of ARS305 and analysis of plasmid mitotic stability identified three short sequence elements within the broad 3'-flanking sequence. The major functional element resides directly 3' of the ACS and the two remaining elements reside further downstream, all within non-conserved ARS sequences. To determine the contribution of the elements to replication origin function in the chromosome, selected linker mutations were transplaced into the ORI305 locus and two-dimensional gel electrophoresis was used to analyze replication bubble formation and fork directions. Mutation of the major functional element identified in the plasmid mitotic stability assay inactivated replication origin function in the chromosome. Mutation of each of the two remaining elements diminished both plasmid ARS and chromosomal origin activities to similar levels. Thus multiple DNA elements identified in the plasmid ARS are determinants of replication origin function in the natural context of the chromosome. Comparison with two other genetically defined chromosomal replicators reveals a conservation of functional elements known to bind ORC, but no two replicators are identical in the arrangement of elements downstream of ORC binding elements or in the extent of functional sequences adjacent to the ACS.

Base Sequence↗
PubMed1997

Origin-specific initiation of mammalian nuclear DNA replication in a Xenopus cell-free system.

The introduction of Chinese hamster ovary (CHO) cell nuclei into Xenopus egg extracts provides the only cell-free system that can efficiently initiate replication at a specific metazoan replication origin. With intact late-G1-phase nuclei as a substrate, the pattern of initiation sites for replication at the CHO dihydrofolate reductase (DHFR) locus is indistinguishable from that observed in cultured cells. By contrast, with early-G1-phase nuclei or with late-G1-phase nuclei that have damaged nuclear envelopes, these same extracts efficiently initiate replication at apparently random sites. Thus, at a distinct point during G1 phase [origin decision point (ODP)], nuclei experience a transition that is required for specific recognition of the DHFR origin by Xenopus egg cytosol. Described here are the basic requirements to achieve origin-specific initiation, which include: 1) a cell line that can be synchronized in G1 phase, 2) a method to prepare intact nuclei, 3) a technique to map origins with a few million cells, and 4) a small colony of Xenopus laevis. Immunodepletion of specific gene products allows one to test hypotheses about the requirements for origin recognition. Here we show that depletion of the Xenopus origin recognition complex subunit XORC2 from Xenopus egg extracts has no influence on the efficiency of replication or the pattern of initiation sites with either pre-ODP or post-ODP nuclei.

Animals↗
PubMed2002

Functional dissection of latency-associated nuclear antigen 1 of Kaposi's sarcoma-associated herpesvirus involved in latent DNA replication and transcription of terminal repeats of the viral genome.

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the maintenance of the viral genome during latent infection. LANA1 colocalizes with KSHV episomes on the host chromosome and mediates their maintenance by attaching these viral structures to host chromosomes. Data from long-term selection of drug resistance in cells conferred by plasmids containing the terminal repeat (TR) sequence of KSHV revealed that KSHV TRs and LANA1 act as cis and trans elements of viral latent replication, respectively. In this study, we further characterized the cis- and trans-acting elements of KSHV latent replication by using a transient replication assay with a methylation-sensitive restriction enzyme, DpnI. Transient reporter and replication assays disclosed that the orientation and basal transcriptional activity of TR constructs did not significantly affect the efficiency of replication. However, at least two TR units were necessary for efficient replication. The N-terminal 90 amino acids comprising the chromosome-binding domain of LANA1 were required for the mediation of LANA1 C-terminal DNA-binding and dimerization domains to support the transient replication of KSHV TRs. LANA1 interacted with components of the origin recognition complexes (ORCs), similar to Epstein-Barr virus nuclear antigen 1. Our data suggest that LANA1 recruits ORCs to KSHV TRs for latent replication of the viral genome.

Antigens, Viral↗
PubMed1999

Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins.

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.

Cell Cycle↗
PubMed1996

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state.

Base Sequence↗
PubMed2006

Essential elements of a licensed, mammalian plasmid origin of DNA synthesis.

We developed a mammalian plasmid replicon with a formerly uncharacterized origin of DNA synthesis, 8xRep*. 8xRep* functions efficiently to support once-per-cell-cycle synthesis of plasmid DNA which initiates within Rep*. By characterizing Rep*'s requirements for acting as an origin, we have uncovered several striking properties it shares with DS, the only other well-characterized, licensed, mammalian plasmid origin of DNA synthesis. Rep* contains a pair of previously unrecognized Epstein-Barr nuclear antigen 1 (EBNA1)-binding sites that are both necessary and sufficient in cis for its origin activity. These sites have an essential 21-bp center-to-center spacing, are bent by EBNA1, and recruit the origin recognition complex. The properties shared between DS and Rep* define cis and trans characteristics of a mammalian, extrachromosomal replicon. The role of EBNA1 likely reflects its evolution from cellular factors involved in the assembly of the initiation machinery.

Base Sequence↗
PubMed1998

Silent chromatin in yeast: an orchestrated medley featuring Sir3p [corrected].

Extensive regions of chromosomes can be transcriptionally repressed through silencing mechanisms mediated by complex chromatin structures. One of the most refined molecular portraits of silenced chromatin comes from studies of the silent mating-type loci and telomeres of S. cerevisiae. In this budding yeast, the Sir3p silent information regulator emerges as a critically important silencing component that interacts with nucleosomes and other silencing proteins. Not only is it essential for silencing, but Sir3p is also capable of spreading silenced chromatin when its dosage is increased. Sir3p is a target of mitogen-activated protein (MAP) kinase cascade regulation and has significant similarity to the Orc1p subunit of the DNA replication origin recognition complex. Thus, in concert with other silencing proteins, Sir3p appears poised to respond to cellular signals and reprogram silencing through replication-associated assembly of repressive chromatin structures.

Chromatin↗
PubMed1999

Chromosomal ARS1 has a single leading strand start site.

Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.

Base Sequence↗
PubMed2002

A approximately 35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP.

BACKGROUND: The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested. RESULTS: Partially purified fractions containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. However, further purification revealed the presence of a putative 35 kDa insect cell protein (p35) which was found responsible for the DNA binding activity. A close match to the 9/11 bases of the ARS consensus sequence was sufficient for p35 binding activity. A DNA fragment from the human c-myc origin region containing yeast ACS like elements also showed p35 binding activity. CONCLUSIONS: We have identified a Spodoptera frugiperda protein with ATP dependent DNA binding activity to ACS like elements. ACS like elements have been reported to be essential for ORC binding and replication initiation in yeast but their role in higher eukaryotes still remains elusive. Like the ARS consensus sequence elements of yeast, ACS like elements found in c-myc and lamin beta 2 origin regions may play similar roles in replication and indicate a conserved role for this DNA motif among eukaryotes.

Adenosine Triphosphate↗
PubMed2002

A novel yeast silencer. the 2mu origin of Saccharomyces cerevisiae has HST3-, MIG1- and SIR-dependent silencing activity.

Silencing in Saccharomyces cerevisiae is found at the mating-type loci HMR and HML, in subtelomeric regions, and at the rDNA locus. Repressed chromatin is built up by the recruitment of the Sir proteins via their interaction with DNA-binding proteins that bind to silencers. Here, we have performed a genetic screen for novel sequence elements within the yeast genome that display silencing activity. We isolated as a novel silencer element the origin of replication from the endogenous 2mu plasmid (2mu ARS). 2mu ARS-mediated silencing was dependent upon the Sir proteins, the origin recognition complex (ORC), and Hst3, a Sir2 histone deacetylase homolog, suggesting that it constituted a novel class of silencing in yeast. Moreover, 2mu ARS carried a binding site for Mig1, a transcriptional repressor of glucose-regulated genes. Both the Mig1-binding site and the MIG1 gene were necessary for full silencing activity of 2mu ARS. Furthermore, Hst3 was physically present at 2mu ARS in a silencing context as well as at the endogenous 2mu plasmid. Also, Hst3 regulated the repression of the flipase gene, although this was likely an indirect effect of HST3 on FLP1 expression.

Fungal Proteins↗