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Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and during chorion gene amplification.

ABSTRACT: The cdc45 protein was originally identified in Saccharomyces cerevisiae and shown to be essential for initiation of eukaryotic DNA replication. Subsequent isolation and characterisation of the corresponding genes from fission yeast, Xenopus and mammals also support a replication role for the protein in these species. They further suggest that during the course of its function cdc45 interacts with a number of other replication proteins, including minichromosome maintenance proteins, the origin recognition complex and DNA polymerase alpha. We have cloned the gene coding for cdc45 protein from Drosophila melanogaster. We have analysed the expression pattern of the cdc45 protein throughout the cell cycle and the life cycle using a combination of indirect immunofluorescence and subcellular fractionation. Our data show that cellular localisation and developmental regulation of the protein is consistent with a role in DNA replication. DmCdc45 is predominantly expressed in proliferating cells. In addition, its subcellular location is nuclear during interphase and the protein shows association with chromatin. The chromatin-associated form of the protein shows a post-translational modification, which may be involved in control of the action of the protein. DmCdc45 shows interactions with mcm proteins, however, the interactions detected show some specificity, perhaps suggesting a preferential association with particular mcm proteins. In addition we show that a stoichiometric mcm interaction may not be obligatory for the function of cdc45 in follicle cell replication, because, unlike the mcm proteins, DmCdc45 localises to the chorion amplification foci in the follicle cells of the ovary.

SUBMITTER: Loebel D

PROVIDER: S-EPMC110785 | biostudies-literature | 2000 Oct

REPOSITORIES: biostudies-literature

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Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and during chorion gene amplification.

Loebel D D Huikeshoven H H Cotterill S S

Nucleic acids research 20001001 20

The cdc45 protein was originally identified in Saccharomyces cerevisiae and shown to be essential for initiation of eukaryotic DNA replication. Subsequent isolation and characterisation of the corresponding genes from fission yeast, Xenopus and mammals also support a replication role for the protein in these species. They further suggest that during the course of its function cdc45 interacts with a number of other replication proteins, including minichromosome maintenance proteins, the origin re ...[more]

PMID: 11024168

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Project description:BackgroundAlthough much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, Schizosaccharomyces pombe.ResultsWe found that over-expressing a non-phosphorylatable form of the replication-initiation protein, Cdc18 (known as Cdc6 in other eukaryotes), drove re-replication of DNA sequences genome-wide, rather than forcing high level amplification of just a few sequences. Moderate variations in extents of re-replication generated regions spanning hundreds of kilobases that were amplified (or not) approximately 2-fold more (or less) than average. However, these regions showed little correlation with replication origins used during S phase. The extents and locations of amplified regions in cells deleted for the checkpoint genes encoding Rad3 (ortholog of human ATR and budding yeast Mec1) and Cds1 (ortholog of human Chk2 and budding yeast Rad53) were similar to those in wild-type cells. Relatively minor but distinct effects, including increased re-replication of heterochromatic regions, were found specifically in cells lacking Rad3. These might be due to Cds1-independent roles for Rad3 in regulating re-replication and/or due to the fact that cells lacking Rad3 continued to divide during re-replication, unlike wild-type cells or cells lacking Cds1. In both wild-type and checkpoint-mutant cells, regions near telomeres were particularly susceptible to re-replication. Highly re-replicated telomere-proximal regions (50-100 kb) were, in each case, followed by some of the least re-replicated DNA in the genome.ConclusionThe origins used, and the extent of replication fork progression, during re-replication are largely independent of the replication and DNA-damage checkpoint pathways mediated by Cds1 and Rad3. The fission yeast pattern of telomere-proximal amplification adjacent to a region of under-replication has also been seen in the distantly-related budding yeast, which suggests that subtelomeric sequences may be a promising place to look for DNA re-replication in other organisms.

Project description:UnlabelledPolydnaviruses are large, double-stranded DNA viruses that are beneficial symbionts of parasitoid wasps. Polydnaviruses in the genus Bracovirus (BVs) persist in wasps as proviruses, and their genomes consist of two functional components referred to as proviral segments and nudivirus-like genes. Prior studies established that the DNA domains where proviral segments reside are amplified during replication and that segments within amplified loci are circularized before packaging into nucleocapsids. One DNA domain where nudivirus-like genes are located is also amplified but never packaged into virions. We recently sequenced the genome of the braconid Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). Here, we took advantage of this resource to characterize the DNAs that are amplified during MdBV replication using a combination of Illumina and Pacific Biosciences sequencing approaches. The results showed that specific nucleotide sites identify the boundaries of amplification for proviral loci. Surprisingly, however, amplification of loci 3, 4, 6, and 8 produced head-to-tail concatemeric intermediates; loci 1, 2, and 5 produced head-to-head/tail-to-tail concatemers; and locus 7 yielded no identified concatemers. Sequence differences at amplification junctions correlated with the types of amplification intermediates the loci produced, while concatemer processing gave rise to the circularized DNAs that are packaged into nucleocapsids. The MdBV nudivirus-like gene cluster was also amplified, albeit more weakly than most proviral loci and with nondiscrete boundaries. Overall, the MdBV genome exhibited three patterns of DNA amplification during replication. Our data also suggest that PacBio sequencing could be useful in studying the replication intermediates produced by other DNA viruses.ImportancePolydnaviruses are of fundamental interest because they provide a novel example of viruses evolving into beneficial symbionts. All polydnaviruses are associated with insects called parasitoid wasps, which are of additional applied interest because many are biological control agents of pest insects. Polydnaviruses in the genus Bracovirus (BVs) evolved ~100 million years ago from an ancestor related to the baculovirus-nudivirus lineage but have also established many novelties due to their symbiotic lifestyle. These include the fact that BVs are transmitted only vertically as proviruses and produce replication-defective virions that package only a portion of the viral genome. Here, we studied Microplitis demolitor bracovirus (MdBV) and report that its genome exhibits three distinct patterns of DNA amplification during replication. We also identify several previously unknown features of BV genomes that correlate with these different amplification patterns.

Dataset Information

Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and during chorion gene amplification.

Publications

Localisation of the DmCdc45 DNA replication factor in the mitotic cycle and during chorion gene amplification.

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