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      The DNA Damage Response Pathway Contributes to the Stability of Chromosome III Derivatives Lacking Efficient Replicators

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          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 5ORIΔ-ΔR 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 5ORIΔ-ΔR 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 5ORIΔ-ΔR 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 ORIΔ 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 ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ 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.

          Author Summary

          Loss of genome integrity underlies aspects of aging and human disease. During DNA replication, two parallel signaling pathways play important roles in the maintenance of genome integrity. One pathway detects DNA damage, while the other senses replication stress. Both pathways activate responses that include arrest of cell cycle progression, giving cells time to cope with the problem. These pathways have been defined by treating cells with compounds that induce either replication stress or DNA damage, but little is known about their roles during unperturbed DNA replication. They may be important when several adjacent replication origins fail to initiate and forks from flanking origins must replicate longer regions of DNA than normal to complete replication. We have used a derivative of budding yeast chromosome III lacking all efficient replication origins to identify mutants that preferentially destabilize this chromosome fragment, which mimics a chromosome with a large inter-origin gap. We found that the DNA damage response pathway, but not the replication stress response pathway, plays an important role in maintaining this fragment. The signal recognized in this case may be replisome failure rather than forks stalled at endogenous DNA damage.

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          Most cited references 91

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          A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
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            A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
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              In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of approximately 4700 deletion mutants. Inviable double-mutant meiotic progeny identify functional relationships between genes. SGA analysis of genes with roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a network of 291 interactions among 204 genes. Systematic application of this approach should produce a global map of gene function.

                Author and article information

                [1 ]Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, United States of America
                [2 ]Banting & Best Department of Medical Research and Department of Molecular Genetics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada
                National Cancer Institute, United States of America
                Author notes

                Conceived and designed the experiments: JFT CI CB CSN. Performed the experiments: JFT CI AD RLB MLT FMdS JYW CSN. Analyzed the data: JFT CI AD RLB CSN. Contributed reagents/materials/analysis tools: CB. Wrote the paper: JFT CB CSN.

                Role: Editor
                PLoS Genet
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                December 2010
                December 2010
                2 December 2010
                : 6
                : 12
                Theis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Pages: 14
                Research Article
                Genetics and Genomics/Chromosome Biology
                Genetics and Genomics/Gene Function
                Genetics and Genomics/Genome Projects
                Molecular Biology/Chromosome Structure
                Molecular Biology/DNA Repair
                Molecular Biology/DNA Replication
                Molecular Biology/Recombination



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