Publications (13) View all
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Article: DNA replication: Pif1 pulls the plug on stalled replication forks.
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ABSTRACT: The conserved PIF helicase family appears to function in replication to ensure termination and passage through regions that slow or arrest replication fork movement. Findings in fission yeast extend evidence from budding yeast, and argue for universal mechanisms that ensure replication integrity.Current biology: CB 05/2012; 22(10):R404-5. · 10.99 Impact Factor -
Article: Nuclear geometry and rapid mitosis ensure asymmetric episome segregation in yeast.
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ABSTRACT: Asymmetric cell division drives the generation of differentiated cells and maintenance of stem cells. In budding yeast, autonomously replicating sequence (ARS) plasmids lacking centromere elements are asymmetrically segregated into the mother cell, where they are thought to contribute to cellular senescence. This phenomenon has been proposed to result from the active retention of plasmids through an interaction with nuclear pores. To investigate the mother-daughter segregation bias of plasmids, we used live-cell imaging to follow the behavior of extrachromosomal DNA. We show that both an excised DNA ring and a centromere-deficient ARS plasmid move freely in the nucleoplasm yet show a strong segregation bias for the mother cell. Computational modeling shows that the geometrical shape of the dividing yeast nucleus and length of mitosis severely restrict the passive diffusion of episomes into daughter nuclei. Predictions based on simulated nuclear division were tested with mutants that extend the length of mitosis. Finally, explaining how various anchors can improve mitotic segregation, we show that plasmid partitioning is improved by tethering the plasmid to segregating structures, such as the nuclear envelope and telomeres. The morphology and brevity of mitotic division in budding yeast impose physical constraints on the diffusion of material into the daughter, obviating the need for a retention mechanism to generate rejuvenated offspring.Current biology: CB 01/2011; 21(1):25-33. · 10.99 Impact Factor -
Article: Actin-related proteins in the nucleus: life beyond chromatin remodelers.
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ABSTRACT: Since their discovery in the mid-1990s, nuclear actin-related proteins (ARPs) have gained attention for their roles as structural components of ATP-dependent chromatin-remodeling complexes. These remodelers can move nucleosomes along the DNA, evict them from chromatin, and exchange histone variants to alter chromatin states locally. Chromatin-remodeling facilitates DNA-templated processes such as transcription regulation, DNA replication, and repair. Consistent with a role for ARPs in shaping chromatin structure, recent genetic studies show that they affect developmental and cell-type specific transcriptional programming. Here, we focus on recent results that suggest a specific contribution of ARPs to long-range interactions in the nucleus, and review evidence indicating that some ARPs may act independently of chromatin-remodeling machines.Current opinion in cell biology 03/2010; 22(3):383-91. · 14.15 Impact Factor -
Article: Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation.
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ABSTRACT: In budding yeast the evolutionarily conserved checkpoint response varies in its sensitivity to DNA damaging agents through the cell cycle. Specifically, higher amounts of damage are needed to activate the downstream checkpoint kinase Rad53 in S-phase cells. We examined here whether phosphorylation of Rad53 itself by cell cycle-dedicated kinases regulates Rad53 activation. We found that during unperturbed growth Rad53 exhibits a small phosphorylation-dependent electrophoretic mobility shift in G(2), M and G(1) phases of the cell cycle that is lost in S phase. We show that Rad53 is phosphorylated in vitro by Cdc5, a mitotic Polo-like kinase, and by the yeast cyclin-dependent kinase, Cdc28. Consistently, the cell cycle-dependent Rad53 mobility shift requires both Cdc5 and Cdc28 activities. We mapped the in vitro targeted phosphorylation sites by mass spectrometry and confirmed with mass spectroscopy that serines 774, 789 and 791 within Rad53 are phosphorylated in vivo in M-phase arrested cells. By creating nonphosphorylatable mutations in the endogenous RAD53 gene, we confirmed that the CDK and Polo kinase target sites are responsible for the observed cell cycle-dependent shift in protein mobility. The loss of phospho-acceptor sites does not interfere with Rad53 activation but accelerates checkpoint adaptation after induction of a single irreparable double-strand break. We thus demonstrate that cell cycle-dependent phosphorylation can fine-tune the response of Rad53 to DNA damage.Cell cycle (Georgetown, Tex.) 01/2010; 9(2):350-63. · 5.36 Impact Factor -
Article: Actin-related protein Arp6 influences H2A.Z-dependent and -independent gene expression and links ribosomal protein genes to nuclear pores.
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ABSTRACT: Actin-related proteins are ubiquitous components of chromatin remodelers and are conserved from yeast to man. We have examined the role of the budding yeast actin-related protein Arp6 in gene expression, both as a component of the SWR1 complex (SWR-C) and in its absence. We mapped Arp6 binding sites along four yeast chromosomes using chromatin immunoprecipitation from wild-type and swr1 deleted (swr1Delta) cells. We find that a majority of Arp6 binding sites coincide with binding sites of Swr1, the catalytic subunit of SWR-C, and with the histone H2A variant Htz1 (H2A.Z) deposited by SWR-C. However, Arp6 binding detected at centromeres, the promoters of ribosomal protein (RP) genes, and some telomeres is independent of Swr1 and Htz1 deposition. Given that RP genes and telomeres both show association with the nuclear periphery, we monitored the ability of Arp6 to mediate the localization of chromatin to nuclear pores. Arp6 binding is sufficient to shift a randomly positioned locus to nuclear periphery, even in a swr1Delta strain. Arp6 is also necessary for the pore association of its targeted RP promoters possibly through cell cycle-dependent factors. Loss of Arp6, but not Htz1, leads to an up-regulation of these RP genes. In contrast, the pore-association of GAL1 correlates with Htz1 deposition, and loss of Arp6 reduces both GAL1 activation and peripheral localization. We conclude that Arp6 functions both together with the nucleosome remodeler Swr1 and also without it, to mediate Htz1-dependent and Htz1-independent binding of chromatin domains to nuclear pores. This association is shown to have modulating effects on gene expression.PLoS Genetics 01/2010; 6(4):e1000910. · 8.69 Impact Factor
