A Screen for Schizosaccharomyces pombe Mutants Defective in Rereplication Identifies New Alleles of rad4+, cut9+ and psf2+

Gómez, Eliana B.; Angeles, Vanessa T.; Forsburg, Susan L.

doi:10.1534/genetics.104.034231

Cited by 19 publications

(29 citation statements)

References 42 publications

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“…This suggests that the fork protection complex is particularly important when MCM helicase activity is abrogated and is consistent with recent work showing replication fork collapse in mcm2ts alleles (Bailis et al 2008). A temperature-sensitive mutant of the GINS subunit psf2 completes a first round of replication at restrictive temperature (Gó mez et al 2005), but is also synthetic lethal with Dswi1 and synthetic sick with Dswi3 (Table 1). In contrast, hsk1-1312, Dswi1, and Dswi3 show little synthetic interaction with replication mutants that arrest prior to replication fork activation: sna41 goa1 /cdc45ts (Uchiyama et al 2001) and pol1-1 (D'Urso et al 1995) ( Table 1).…”

Section: Resultssupporting

confidence: 89%

Fission Yeast Hsk1 (Cdc7) Kinase Is Required After Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage

Dolan

Schmidt

et al. 2010

Genetics

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Genome stability in fission yeast requires the conserved S-phase kinase Hsk1 (Cdc7) and its partner Dfp1 (Dbf4). In addition to their established function in the initiation of DNA replication, we show that these proteins are important in maintaining genome integrity later in S phase and G2. hsk1 cells suffer increased rates of mitotic recombination and require recombination proteins for survival. Both hsk1 and dfp1 mutants are acutely sensitive to alkylation damage yet defective in induced mutagenesis. Hsk1 and Dfp1 are associated with the chromatin even after S phase, and normal response to MMS damage correlates with the maintenance of intact Dfp1 on chromatin. A screen for MMS-sensitive mutants identified a novel truncation allele, rad35 (dfp1-(1-519)), as well as alleles of other damage-associated genes. Although Hsk1-Dfp1 functions with the Swi1-Swi3 fork protection complex, it also acts independently of the FPC to promote DNA repair. We conclude that Hsk1-Dfp1 kinase functions post-initiation to maintain replication fork stability, an activity potentially mediated by the C terminus of Dfp1.T HE Hsk1 protein kinase, the fission yeast ortholog of Saccharomyces cerevisiae Cdc7, is a conserved protein essential for the initiation of DNA replication (Masai et al. 1995;Brown and Kelly 1998;Snaith et al. 2000). Data from many systems suggest that the kinase functions at individual replication origins to activate the prereplication complex (preRC) through phosphorylation of the MCM helicase and other subunits (reviewed in Forsburg 2004). In fission yeast, Hsk1 kinase activity is limited to S phase by its regulatory subunit Dfp1, which is transcriptionally and posttranslationally regulated to restrict its peak of activity to S phase (Brown and Kelly 1999;Takeda et al. 1999). The requirement for Dfp1 (in S. cerevisiae, Dbf4) is similar to the dependence of CDK kinases on cyclin activity; thus, the Ccd7 kinase family has been dubbed DDK (Dbf4-dependent kinases) ( Johnston et al. 1999;Duncker and Brown 2003). Hsk1 is a target of the Cds1 checkpoint kinase and undergoes Cds1-dependent phosphorylation during hydroxyurea (HU) treatment in vivo and in vitro (Snaith et al. 2000). Interestingly, deletion of Dcds1 partly rescues hsk1-1312 temperature sensitivity, which suggests that Hsk1 is negatively regulated by the replication checkpoint. In turn, Cds1 is poorly activated in hsk1 mutants after HU treatment, indicating that there may be a feedback loop linking these two kinases (Snaith et al. 2000;Takeda et al. 2001). hsk1 mutants are sensitive to HU treatment, with a phenotype suggesting a specific defect in recovery (Snaith et al. 2000).DDK kinases have substrates outside of the replication initiation pathway. Functional dissection of Schizosaccaromyces pombe Dfp1 identifies separate regions that are required for checkpoint response (N-terminal domain; Takeda et al. 1999;Fung et al. 2002), for centromere cohesion and replication (MIR domain;Bailis et al. 2003;Hayashi et al. 2009) and for proper response to alkylat...

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Section: Resultssupporting

confidence: 89%

Fission Yeast Hsk1 (Cdc7) Kinase Is Required After Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage

Dolan

Schmidt

et al. 2010

Genetics

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“…Arg2p and Ura5p are involved in arginine and uracil synthesis, respectively. During the course of this study, we independently identified bsh3 ϩ as psf2 ϩ (SPBC725.13c), the homolog of budding yeast PSF2 (16). Deletion of S. pombe bsh3 ϩ /psf2 ϩ from the complementing library DNA fragment abolished rescue of the bir1-46 growth defect at 34°C (data not shown), indicating that psf2 ϩ is a high-copy-number suppressor of bir1-46.…”

Section: Resultsmentioning

confidence: 99%

“…(AK014776) and Caenorhabditis elegans (F31C3.5). The S. cerevisiae and Xenopus Psf2p proteins, as well as S. pombe Psf2p, are known to affect DNA replication (16,27,30,51).…”

Section: Resultsmentioning

confidence: 99%

“…We independently identified fission yeast Psf2p in a screen for mutants that block rereplication (16). Psf2p homologs in Saccharomyces cerevisiae and Xenopus laevis were recently described as components of the GINS protein complex, which consists of four subunits (Sld5p/Cdc105p, Psf1p/Cdc101p, Psf2p/Cdc102p, and Psf3p) and is essential for DNA replication (27,30,51).…”

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confidence: 99%

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Suppressors of Bir1p (Survivin) Identify Roles for the Chromosomal Passenger Protein Pic1p (INCENP) and the Replication Initiation Factor Psf2p in Chromosome Segregation

Huang

Bailis

Leverson

et al. 2005

Molecular and Cellular Biology

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Fission yeast Bir1p/Cut17p/Pbh1p, the homolog of human Survivin, is a conserved chromosomal passenger protein that is required for cell division and cytokinesis. To study how Bir1p promotes accurate segregation of chromosomes, we generated and analyzed a temperature-sensitive allele, bir1-46, and carried out genetic screens to find genes that interact with bir1 ؉ . We identified Psf2p, a component of the GINS complex required for DNA replication initiation, as a high-copy-number suppressor of the bir1-46 growth defect. Loss of Psf2p function by depletion or deletion or by use of a temperature-sensitive allele, psf2-209, resulted in chromosome missegregation that was associated with mislocalization of Bir1p. We also found that the human homolog of Psf2p, PSF2, was required for proper chromosome segregation. In addition, we observed that high-copynumber expression of Pic1p, the fission yeast homolog of INCENP (inner centromere protein), suppressed bir1-46. Pic1p exhibited a localization pattern typical of chromosomal passenger proteins. Deletion of pic1 ؉ caused chromosome missegregation phenotypes similar to those of bir1-46. Our data suggest that Bir1p and Pic1p act as part of a conserved chromosomal passenger complex and that Psf2p/GINS indirectly affects the localization and function of this complex in chromosome segregation, perhaps through an S-phase role in centromere replication.

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“…A total of nine different ts mutations have been identified in the four yeast GINS proteins (8,17,18). These mutated residues are highlighted in the sequence alignments shown in SI Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the GINS complex and functional insights into its role in DNA replication

Chang

Wang

Bermudez

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

120

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The GINS complex, which contains the four subunits Sld5, Psf1, Psf2, and Psf3, is essential for both the initiation and progression of DNA replication in eukaryotes. GINS associates with the MCM2-7 complex and Cdc45 to activate the eukaryotic minichromosome maintenance helicase. It also appears to interact with and stimulate the polymerase activities of DNA polymerase and the DNA polymerase ␣-primase complex. To further understand the functional role of GINS, we determined the crystal structure of the full-length human GINS heterotetramer. Each of the four subunits has a major domain composed of an ␣-helical bundle-like structure. With the exception of Psf1, each of the other subunits has a small domain containing a three-stranded ␤-sheet core. Each full-length protein in the crystal has unstructured regions that are all located on the surface of GINS and are probably involved in its interaction with other replication factors. The four subunits contact each other mainly through ␣-helices to form a ring-like tetramer with a central pore. This pore is partially plugged by a 16-residue peptide from the Psf3 N terminus, which is unique to some eukaryotic Psf3 proteins and is not required for tetramer formation. Removal of these N-terminal 16 residues of Psf3 from the GINS tetramer increases the opening of the pore by 80%, suggesting a mechanism by which accessibility to the pore may be regulated. The structural data presented here indicate that the GINS tetramer is a highly stable complex with multiple flexible surface regions.Cdc45 ͉ DNA helicase ͉ minichromosome maintenance complex ͉ DNA polymerase E ukaryotic DNA replication is controlled by a series of ordered and regulated steps (1-3) that commence with the binding of the six-subunit origin recognition complex (ORC) to replication origins. During the G1 phase of the cell cycle, Cdc6, and Cdt1 are recruited to the origin, and together with ORC, support the loading of the heterohexameric MCM2-7 complex (minichromosome maintenance, MCM) to form the prereplication complex (pre-RC). Although a substantial amount of data suggest that MCM acts as the replicative helicase, MCM present in the pre-RC (as well as isolated MCM) is devoid of helicase activity (summarized in ref. 4). At the G 1 /S transition of the cell cycle, it appears that the MCM helicase activity is activated by a complex and an as yet poorly understood series of modifications that require the action of two protein kinases, DDK (Cdc7-Dbf4) and CDK (cyclin-dependent), as well as the participation of at least eight additional factors, including Mcm10, Cdc45, Dpb11, GINS, synthetic lethal with dpb11 mutant-2 (Sld2), and Sld3 (4). Two of these components, Cdc45 and GINS, appear critical for helicase activation because DNA unwinding is observed (3, 5), concomitant with their loading at origins. In accord with these findings, a complex containing nearstoichiometric levels of MCM, Cdc45, and GINS was isolated from Drosophila and shown to possess DNA helicase activity (6). Studies with Xenopus extracts revealed...

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A Screen for Schizosaccharomyces pombe Mutants Defective in Rereplication Identifies New Alleles of rad4+, cut9+ and psf2+

Cited by 19 publications

References 42 publications

Fission Yeast Hsk1 (Cdc7) Kinase Is Required After Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage

Fission Yeast Hsk1 (Cdc7) Kinase Is Required After Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage

Suppressors of Bir1p (Survivin) Identify Roles for the Chromosomal Passenger Protein Pic1p (INCENP) and the Replication Initiation Factor Psf2p in Chromosome Segregation

Crystal structure of the GINS complex and functional insights into its role in DNA replication

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