Dataset Information

The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks.

ABSTRACT: The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication. Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast. We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue (Orp4p) consists of two distinct functional domains. The C-terminal domain shows strong sequence similarity to human, frog, and yeast Orc4 proteins, including conserved ATP-binding motifs. The N-terminal domain contains nine copies of the AT-hook motif found in a number of DNA-binding proteins, including the members of the HMG-I(Y) family of chromatin proteins. AT-hook motifs are known from biochemical and structural studies to mediate binding to the minor groove of AT-tracts in DNA. Orp4p is essential for viability of Sc. pombe and is expressed throughout the cell cycle. The Orp4 protein (and its isolated N-terminal domain) binds to the Sc. pombe replication origin, ars1. The DNA binding properties of Orp4p provide a plausible explanation for the characteristic features of Sc. pombe origins of replication, which differ significantly from those of Sa. cerevisiae.

SUBMITTER: Chuang RY

PROVIDER: S-EPMC15824 | biostudies-literature | 1999 Mar

REPOSITORIES: biostudies-literature

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The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks.

Chuang R Y RY Kelly T J TJ

Proceedings of the National Academy of Sciences of the United States of America 19990301 6

The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication. Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast. We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue (Orp4p) consists of two disti ...[more]

PMID: 10077566

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Project description:Deoxyribonucleic acid replication is one of the most crucial tasks taking place in the cell, and it has to be precisely regulated. This process is initiated in the replication origins (ORIs), and thus it is essential to identify such sites for a deeper understanding of the cellular processes and functions related to the regulation of gene expression. Considering the important tasks performed by ORIs, several experimental and computational approaches have been developed in the prediction of such sites. However, existing computational predictors for ORIs have certain curbs, such as building only single-feature encoding models, limited systematic feature engineering efforts and failure to validate model robustness. Hence, we developed a novel species-specific yeast predictor called yORIpred that accurately identify ORIs in the yeast genomes. To develop yORIpred, we first constructed optimal 40 baseline models by exploring eight different sequence-based encodings and five different machine learning classifiers. Subsequently, the predicted probability of 40 models was considered as the novel feature vector and carried out iterative feature learning approach independently using five different classifiers. Our systematic analysis revealed that the feature representation learned by the support vector machine algorithm (yORIpred) could well discriminate the distribution characteristics between ORIs and non-ORIs when compared with the other four algorithms. Comprehensive benchmarking experiments showed that yORIpred achieved superior and stable performance when compared with the existing predictors on the same training datasets. Furthermore, independent evaluation showcased the best and accurate performance of yORIpred thus underscoring the significance of iterative feature representation. To facilitate the users in obtaining their desired results without undergoing any mathematical, statistical or computational hassles, we developed a web server for the yORIpred predictor, which is available at: http://thegleelab.org/yORIpred.

Project description:BackgroundIn budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2).ResultsOur microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (approximately 3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells.ConclusionThe fact that approximately 97% of fission yeast replication origins - both early and late - are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.

Dataset Information

The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks.

Publications

The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks.

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