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Nuclear myosin VI maintains replication fork stability

ABSTRACT: The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous (F-) actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase WRNIP1 in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized DARPins to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Hela Cell

SUBMITTER: Petra Beli

LAB HEAD: Petra Beli

PROVIDER: PXD035394 | Pride | 2023-05-04

REPOSITORIES: Pride

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			Action	DRS
	20160708_QEP1_PBE_PB_sc_silac_gst_myosin_wt_i1104a_s1_ingel_01.raw	Raw
	20160708_QEP1_PBE_PB_sc_silac_gst_myosin_wt_i1104a_s1_ingel_02.raw	Raw
	20160708_QEP1_PBE_PB_sc_silac_gst_myosin_wt_i1104a_s1_ingel_03.raw	Raw
	20160708_QEP1_PBE_PB_sc_silac_gst_myosin_wt_i1104a_s1_ingel_04.raw	Raw
	20160708_QEP1_PBE_PB_sc_silac_gst_myosin_wt_i1104a_s1_ingel_05.raw	Raw

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Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip • The goal of the experiment Genome-wide localization of Ino80 on chromosome in Saccharomyces cerevisiae • Keywords DNA replication, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI) • Experimental factor Distribution of Ino80 in random culture Distribution of Ino80 in G1 phase Distribution of Ino80 in early S phase • Experimental design ChIP analyses: W303 background cells expressing Myc-tagged Ino80 were used for the ChIP using anti-Myc monoclonal antibody (9E11). ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI) was used. • Quality control steps taken Confirmation of several loci by quantitative real time PCR.

Project description:Background: Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double strand breaks, but also to those that impair replication fork progression. Results: To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the Ino80 chromatin remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the presence of the Ino80 complex at stalled forks and at unfired origins increased dramatically. Importantly, the resumption of DNA replication after release from a HU block was impaired in the absence of Ino80 activity. Mutant cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. Conclusions: The Ino80 chromatin remodeling complex is enriched at stalled replication forks where it promotes the resumption of replication upon recovery from fork arrest. Keywords: ChIP-chip • The goal of the experiment Genome-wide localization of Ino80 and Arp5 on chromosome in Saccharomyces cerevisiae • Keywords DNA replication, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI) • Experimental factor Distribution of Ino80 and Arp5 in wild type in random culture Distribution of Ino80 in G1 cells Distribution of Ino80 in early S phase cells • Experimental design ChIP analyses: W303 background cells expressing Myc tagged Ino80 were used for the ChIP using anti-Myc monoclonal antibody (9E11). ChIP analyses: W303 background cells expressing Myc tagged Ino80 were used for the ChIP using anti-Arp5 polyclonal antibody. ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI) was used. • Quality control steps taken Confirmation of several loci by quantitative real time PCR.

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Nuclear myosin VI maintains replication fork stability

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