Comment[ArrayExpressAccession] E-GEOD-46841 MAGE-TAB Version 1.1 Public Release Date 2013-09-20 Investigation Title DNA replication and DSB factor binding in the meiotic S-phase checkpoint in budding yeast Comment[Submitted Name] DNA replication and DSB factor binding in the meiotic S-phase checkpoint in budding yeast Experiment Description During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR-dependent replication checkpoint in budding yeast that prevented the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint suppressed DSBs through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis. DSB factor association was measured in wild-type and checkpoint mutants strains under non-inducing or replication checkpoint inducing conditions. Additionally, DNA replication and helicase loading were measured in a replication and checkpoint deficient strain (cdc6-mn). Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Hochwagen Blitzblau Hochwagen Person First Name Andreas Hannah Andreas Person Mid Initials G Person Email andi@nyu.edu Person Affiliation New York University Person Address New York University, 1009 Silver Center 100 Washington Square East, New York, NY, USA Person Roles submitter Protocol Name P-GSE46841-1 P-GSE46841-2 P-GSE46841-10 P-GSE46841-5 P-GSE46841-6 P-GSE46841-8 P-GSE46841-3 P-GSE46841-9 P-GSE46841-4 P-GSE46841-7 Protocol Description Cy3 and Cy5 levels and background were calculated using Agilent Feature Extractor software. Background normalization, log2 ratios for each experiment and scale normalizations across each set of duplicated experiments were calculated using the sma package in R (Yang, et al., 2001), a computer language and environment for statistical computing (v2.1.0, http://www.r-project.org). ID_REF = VALUE = scale normalized log2 ratio of experimental DNA over control DNA sample Cy3 and Cy5 levels and background were calculated using Agilent Feature Extractor software. Background normalization, log2 ratios for each experiment and scale normalizations across each set of duplicated experiments were calculated using the sma package in R (Yang, et al., 2001), a computer language and environment for statistical computing (v2.1.0, http://www.r-project.org). ID_REF = VALUE = Replicate datasets are the normalized log2 ratio (8h gDNA / control G1 unreplicated DNA). The final values shown in the paper are average of the individual replicate log2 ratios. not provided Half of the immunoprecipitated DNA or one tenth of the input DNA was labeled with Cy3- or Cy5-dUTP (GE Healthcare) by random priming using 4 µg random nonamer oligo (Integrated DNA Technologies) and 10 units of Klenow (New England Biolabs). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore). Samples were co-hybridized to custom Agilent arrays in 1X Agilent hybridization buffer at 65 degrees for 16 hours. Samples were washed according to manufacturer's instructions. Cells were pre-grown in presporulation medium (BYTA) and transferred into sporulation medium (SPO) at 0 hours. Samples were isolated after 8 hours. Control DNA samples were isolated from G1 haploid SK1, which were incubated for 3 hours with 5 μg/ml alpha-factor. Cells were pre-grown in presporulation medium (BYTA) and transferred into sporulation medium (SPO) at 0 hours. Cells were lysed by bead beating in 500 µl phenol/chloroform and 500 µl of breakage buffer (10 mM TRIS, pH 8.0, 1 mM EDTA, 100 mM NaCl, 2% Triton X-100, 1% SDS). After centrifugation, the aqueous phase was precipitated with ethanol and resuspended in 500 µl TE7.6 (10 mM TRIS, pH7.6, 1 mM EDTA) with 30 µg RNase. DNA was resuspended and samples were incubated at 50°C for 3 hours. DNA was sheared by sonicating at 100% output and lowest power for 10 seconds using a Branson sonicator. DNA was extracted with 500 µl phenol/chloroform, precipitated with ethanol and resuspended in 100 µl of TE7.6 25 ml of cells were harvested after 30 minutes (Mcm2-7) or 3 hours (all other experiment) in sporulation media. Chromatin immunoprecipitation was performed as described (Aparicio et al., 1997). One tenth of the lysate was removed as an input sample. Proteins were immunoprecipitated with the indicated antibodies in combination with 20 µl GammaBind-Sepharose (GE Healthcare) for 16 hours at 4°C. Agilent scanner using the AgilentHD_CGH protocol (5μM resolution, 100% PMT for each laser). Images were quantified using Agilent Feature Extraction Software (version 10.1.1.1). Protocol Type normalization data transformation protocol normalization data transformation protocol labelling protocol labelling protocol hybridization protocol growth protocol growth protocol nucleic acid extraction protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name STRAIN OR LINE TIME IN SPO HOURS CHIP ANTIBODY GENOTYPE Experimental Factor Type strain or line time in spo hours chip antibody genotype Comment[SecondaryAccession] GSE46841 Comment[GEOReleaseDate] 2013-09-20 Comment[ArrayExpressSubmissionDate] 2013-05-10 Comment[GEOLastUpdateDate] 2013-09-20 Comment[AEExperimentType] ChIP-chip by tiling array Comment[AEExperimentType] comparative genomic hybridization by array SDRF File E-GEOD-46841.sdrf.txt