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Results for “Origin Recognition Complex”

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PubMed2003

Lack of SeqA focus formation, specific DNA binding and proper protein multimerization in the Escherichia coli sequestration mutant seqA2.

In Escherichia coli wild-type cells newly formed origins cannot be reinitiated. The prevention of reinitiation is termed sequestration and is dependent on the hemimethylated state of newly replicated DNA. Several mutants discovered in a screen for the inability to sequester hemimethylated origins have been mapped to the seqA gene. Here, one of these mutants, seqA2, harbouring a single amino acid change in the C-terminal end of the SeqA protein, was found to also be unable to form foci in vivo. The SeqA foci seen in the wild-type cells are believed to arise from multimerization of SeqA on hemimethylated DNA at the replication fork, presumably representing organization of newly formed DNA by SeqA. The result suggests that the process of origin sequestration is closely tied to the process of focus maintenance at the replication fork. In vitro, purified SeqA2 protein was found incapable of forming highly ordered multimers that bind hemimethylated oriC. The mutant protein was also incapable of restraining negative supercoils. Both in vivo and in vitro results support the idea that origin sequestration is an integral part of organization of newly formed DNA performed by SeqA.

Bacterial Outer Membrane Proteins↗
PubMed2003

Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors, elongation factors, and origins.

The integrity of chromosomes during cell division is ensured by both trans-acting factors and cis-acting chromosomal sites. Failure of either these chromosome integrity determinants (CIDs) can cause chromosomes to be broken and subsequently misrepaired to form gross chromosomal rearrangements (GCRs). We developed a simple and rapid assay for GCRs, exploiting yeast artificial chromosomes (YACs) in Saccharomyces cerevisiae. We used this assay to screen a genome-wide pool of mutants for elevated rates of GCR. The analyses of these mutants define new CIDs (Orc3p, Orc5p, and Ycs4p) and new pathways required for chromosome integrity in DNA replication elongation (Dpb11p), DNA replication initiation (Orc3p and Orc5p), and mitotic condensation (Ycs4p). We show that the chromosome integrity function of Orc5p is associated with its ATP-binding motif and is distinct from its function in controlling the efficiency of initiation of DNA replication. Finally, we used our YAC assay to assess the interplay of trans and cis factors in chromosome integrity. Increasing the number of origins on a YAC suppresses GCR formation in our dpb11 mutant but enhances it in our orc mutants. This result provides potential insights into the counterbalancing selective pressures necessary for the evolution of origin density on chromosomes.

Cell Cycle Proteins↗
PubMed2006

Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA.

Regions of bacterial chromosomes occupy characteristic locations within the cell. In Bacillus subtilis, the origin of replication, oriC, is located at 0 degrees /360 degrees on the circular chromosome. After duplication, sister 0 degrees regions rapidly move to and then reside near the cell quarters. It has been hypothesized that origin function or oriC sequences contribute to positioning and movement of the 0 degrees region. We found that the position of a given chromosomal region does not depend on initiation of replication from the 0 degrees region. In an oriC mutant strain that replicates from a heterologous origin (oriN) at 257 degrees , the position of both the 0 degrees and 257 degrees regions was similar to that in wild-type cells. Thus, positioning of chromosomal regions appears to be independent of which region is replicated first. Furthermore, we found that neither oriC sequences nor the replication initiator DnaA is required or sufficient for positioning a region near the cell quarters. A sequence within oriC previously proposed to play a critical role in chromosome positioning and partitioning was found to make little, if any, contribution. We propose that uncharacterized sites outside of oriC are involved in moving and/or maintaining the 0 degrees region near the cell quarters.

Bacillus subtilis↗
PubMed2001

DNA replication. Genomic views of genome duplication.

DNA replication is initiated at numerous origins of replication (oris) within the chromosomes. In a pair of ambitious studies, two groups have used different techniques to pinpoint the locations of all of the oris throughout the yeast genome at different times during S phase (Raghuraman et al., Wyrick et al.). Stillman, in his Perspective, compares and contrasts the different methods and their findings, and speculates on the value of combining these techniques to look at oris in the human genome.

Binding Sites↗
PubMed2002

Conservation of the COP9/signalosome in budding yeast.

BACKGROUND: The COP9/signalosome (CSN), a multiprotein complex consisting of eight subunits, is implicated in a wide variety of regulatory processes including cell cycle control, signal transduction, transcriptional activation, and plant photomorphogenesis. Some of these functions have been linked to CSN-associated enzymes, including kinases and an activity that removes the ubiquitin-like protein NEDD8/Rub1p from the cullin subunit of E3 ligases. CSN is highly conserved across species from fission yeast to humans, but sequence comparison has failed to identify the complex in budding yeast, except for a putative CSN5 subunit called Rri1p. RESULTS: We show that disruption of four budding yeast genes, PCI8 and three previously uncharacterized ORFs, which encode proteins interacting with Rrr1p/Csn5p, each results in the accumulation of the cullin Cdc53p exclusively in the Rub1p-modified state. This phenotype, which resembles that of fission yeast csn mutants, is due to a biochemical defect in deneddylation that is complemented by wild-type cell lysate and by purified human CSN in vitro. Although three of the four genes encode proteins with PCI domains conserved in metazoan CSN proteins, their disruption does not confer the DNA damage sensitivity described in some fission yeast csn mutants. CONCLUSIONS: Our studies present unexpected evidence for the conservation of a functional homologue of the metazoan CSN, which mediates control of cullin neddylation in budding yeast.

Animals↗
PubMed2006

Genome-wide hierarchy of replication origin usage in Saccharomyces cerevisiae.

Replication origins in a genome are inherently different in their base sequence and in their response to temporal and cell cycle regulation signals for DNA replication. To investigate the chromosomal determinants that influence the efficiency of initiation of DNA replication genome-wide, we made use of a reverse strategy originally used for the isolation of replication initiation mutants in Saccharomyces cerevisiae. In yeast, replication origins isolated from chromosomes support the autonomous replication of plasmids. These replication origins, whether in the context of a chromosome or a plasmid, will initiate efficiently in wild-type cells but show a dramatically contrasted efficiency of activation in mutants defective in the early steps of replication initiation. Serial passages of a genomic library of autonomously replicating sequences (ARSs) in such a mutant allowed us to select for constitutively active ARSs. We found a hierarchy of preferential initiation of ARSs that correlates with local transcription patterns. This preferential usage is enhanced in mutants defective in the assembly of the prereplication complex (pre-RC) but not in mutants defective in the activation of the pre-RC. Our findings are consistent with an interference of local transcription with the assembly of the pre-RC at a majority of replication origins.

DNA, Fungal↗
PubMed2003

Is DNA sequence sufficient to specify DNA replication origins in metazoan cells?

DNA replication occupies a central position in the cell cycle and, therefore, in the development and life of multicellular organisms. During the last 10 years, our comprehension of this important process has considerably improved. Although the mechanisms that coordinate DNA replication with the other moments of the cell cycle are not yet fully understood, it is known that they mainly operate through DNA replication origins and the protein complexes bound to them. In eukaryotes, the packaging status of chromatin seems to be part of the mechanism that controls whether or not and when during the S-phase a particular origin will be activated. Intriguingly, the protein complexes bound to DNA replication origins appear to be directly involved in controlling chromatin packaging. In this manner they can also affect gene expression. In this review we focus on DNA replication origins in metazoan cells and on the relationship between these elements and the structural and functional organization of the genome.

Animals↗
PubMed2000

Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei.

As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G(1)-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-beta) and (beta') that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-(beta/(beta)' appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G(1)-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G(1)-phase. Third, selective activation of ori-(beta) in late G(1)-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.

Animals↗
PubMed2006

Regulating the licensing of DNA replication origins in metazoa.

Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins ('DNA licensing') is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states.

Animals↗
PubMed2003

Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro.

BACKGROUND: The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA. RESULTS: A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced. CONCLUSIONS: The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.

Base Sequence↗
PubMed2000

Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18.

We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.

Anaphase↗
PubMed1996

The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase.

In a screen for new cell-cycle genes in Schizosaccharomyces pombe we have isolated cdc30, which is identical to orp1, a putative homolog of the Saccharomyces cerevisiae ORC1 gene. Analysis of the temperature-sensitive orp1-4 and the orp1(delta) mutants indicates that orp1 is required at the onset of S phase for an early step of DNA replication. Orp1p is found in the nucleus and is present at a constant level throughout the cell cycle. Genetic interactions occur between orp1 and cdc18 and cdc21 (an MCM homolog). Orp1p forms protein complexes with both cdc18p and cdc21p in vivo, suggesting that interactions between these proteins and ORC are important for controlling the initiation of DNA replication at the onset of S phase. The orp1 gene is also required for the control that prevents entry into mitosis in the absence of DNA replication, suggesting a role for ORC in this checkpoint pathway.

Cell Cycle↗
PubMed2006

Stringent and relaxed recognition of oriT by related systems for plasmid mobilization: implications for horizontal gene transfer.

The plasmids R1162 and pSC101 have origins of conjugative transfer (oriTs) and corresponding relaxases that are closely related. The oriTs are made up of a highly conserved core, where DNA is cleaved by the relaxase prior to transfer, and an inverted repeat that differs in size and sequence. We show that in each case the seven base pairs adjacent to the core and within one arm of the inverted repeat are sufficient to determine specificity. Within this DNA there are three AT base pairs located 4 bp from the core. Mutations in the AT base pairs suggest that the relaxase makes essential contacts at these locations to the minor groove of the DNA. The remaining four bases are different for each oriT and are both necessary and sufficient for stringent recognition of oriT by the pSC101 mobilization proteins. In contrast, the R1162 mobilization proteins have a much more relaxed requirement for the base sequence of this specificity region. As a result, the R1162 mobilization proteins can initiate transfer from a variety of sites, including those derived from the chromosome. The R1162 mobilization proteins could therefore contribute to the horizontal gene transfer of DNA from diverse sources.

Bacterial Proteins↗
PubMed2004

Developmental gene amplification and origin regulation.

Developmentally regulated gene amplification serves to increase the number of templates for transcription, yielding greatly increased protein and/or RNA product for gene(s) at the amplified loci. It is observed with genes that are very actively transcribed and during narrow windows of developmental time where copious amounts of those particular gene products are required. Amplification results from repeated firing of origins at a few genomic loci, while the rest of the genome either does not replicate, or replicates to a lesser extent. As such, amplification is a striking exception to the once-and-only-once rule of DNA replication and may be informative as to that mechanism. Drosophila amplifies eggshell (chorion) genes in the follicle cells of the ovary to allow for rapid eggshell synthesis. Sciara amplifies multiple genes in larval salivary gland cells that encode proteins secreted in the saliva for the pupal case. Finally, Tetrahymena amplifies its rRNA genes several thousand-fold in the creation of the transcriptionally active macronucleus. Due to the ease of molecular and genetic analysis with these systems, the study of origin regulation has advanced rapidly. Comparisons reveal an evolutionarily conserved trans-regulatory apparatus and a similar organization of sequence-specific cis-regulatory replicator and origin elements. The studies indicate a regulatory role for chromatin structure and transcriptionally active genes near the origins.

Animals↗