Dataset Information

Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site.

ABSTRACT: Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.

SUBMITTER: Chang F

PROVIDER: S-EPMC2519699 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site.

Chang Fujung F Theis James F JF Miller Jeremy J Nieduszynski Conrad A CA Newlon Carol S CS Weinreich Michael M

Molecular and cellular biology 20080623 16

Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to ...[more]

PMID: 18573888

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Project description:In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORI?-?R fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective mrc1(AQ) allele did not affect 5ORI?-?R fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORI?-?R fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORI? chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORI? chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORI? chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps.

Dataset Information

Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site.

Publications

Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site.

Similar Datasets

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