Comment[ArrayExpressAccession] E-GEOD-43384 MAGE-TAB Version 1.1 Public Release Date 2013-06-01 Investigation Title Budding yeast ATM/ATR contribute to meiotic double-strand-break (DSB) homeostasis by down-regulating Rec114, an essential component of the DSB-machinery Comment[Submitted Name] Budding yeast ATM/ATR contribute to meiotic double-strand-break (DSB) homeostasis by down-regulating Rec114, an essential component of the DSB-machinery Experiment Description In most organisms, meiotic recombination begins with programmed DNA double strand break (DSB) formation by Spo11. Here, we present evidence that Tel1/Mec1, the budding yeast ATM/ATR, regulate DSB formation by phosphorylating Rec114, an essential Spo11-accessory protein. Analyses of a non-phosphorylatable- or phosphomimetic- alleles of rec114 revealed that DSB-dependent phosphorylation of Rec114 limited its association with DSB-hotspots resulting in reduction in DSB formation. Also observed were the impact of Rec114 phosphorylation on its homolog synapsis-associated removal from chromosomes and NDT80-dependent turnover. Specifically, we found that the synapsis- and NDT80-dependent Rec114 downregulation occurred later in the rec114 mutant with a reduced Spo11-catalysis, but earlier in the other with an enhanced catalysis, strongly implicating the existence of a feedback mechanism coupling the extent of Spo11-catalysis to Rec114 activity. Taken together, these observations suggest that three different mechanisms of down regulating Rec114 contribute to meiotic DSB homeostasis, a feedback mechanism to maintain the number of meiotic DSBs at the developmentally programmed level. 6 genome wide ChIPchip sets: 3 for meiotic DSB formation (Spo11-ChIP) and 3 for protein-DNA association (Rec114-ChIP), each for wild type and two mutants during meiosis (corresponding to the main Figure 3, as well as to Figures S3, S4, S5). 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 Klein Carballo Panizza Serrentinoc L. Johnsona Geyomat Borde Klein Cha Person First Name Franz Jesus Silvia Maria-Elisabetta Anthony Marco Valerie Franz Rita Person Email franz.klein@univie.ac.at Person Affiliation University of Vienna, MFPL Person Phone ++431427756220 Person Fax ++43142779562 Person Address Chromosome Biology, University of Vienna, MFPL, Dr. Bohrg. 1, Vienna, Austria Person Roles submitter Protocol Name P-GSE43384-3 P-GSE43384-4 P-GSE43384-1 P-GSE43384-2 P-GSE43384-5 Protocol Description Two rounds of amplification, DNA fragmentation, and end-labeling, were performed according to the Affymetrix ChIP protocol. Hybridization to Affymetrix S. cerevisiae Tiling 1.0R Arrays was carried out at 42C for 16 hours. The following washing and staining steps were done with an Affymetrix 450 station. All reagents used are described in the Affymetrix ChIP Assay Protocol. Meiotic cultures of diploid S. cerevisiae SK1 strain were set up as follows: A saturated YPD culture was used to inoculate 300 ml liquid SPS medium (.5% yeast extract, 1% Bacto peptone, 1% potassium acetate, .17% Yeast nitrgen base w/o aa, 0.05 M potassium biphtalate, pH 5.5) 1:400 and grown in a 3-liter baffled flask at 200 rpm at 30°C for 14 hrs. Cells were harvested, washed once, resuspended at an OD660 of 1.1 in sporulation medium (1% potassium acetate pH 7.0, supplemented with amino acids and .001% PPG) and shaken at 200 rpm at 30°C for the appropriate time. Except for the samples labelled "-FA", where formaldehyde was omitted, cells were fixed in 1% formaldehyde for 30min. 1X10^9 cells were crushed by glassbeads using a MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka). Chromosomal DNA was sheared to an average size of 500-1000bp by sonication. Chromatin IP was performed as described in Katou et al. (Methods Enzym., 409,389-410, 2006). Each array was scanned by Genechip Scanner 3000 (Affymetrix, CA) at default settings for this type of array. Protocol Type labelling protocol hybridization protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name MEIOTIC TIMEPOINT GENOTYPE BAIT PROTEIN ANTIBODY Experimental Factor Type meiotic timepoint genotype bait protein antibody Comment[SecondaryAccession] GSE43384 Comment[GEOReleaseDate] 2013-06-01 Comment[ArrayExpressSubmissionDate] 2013-01-09 Comment[GEOLastUpdateDate] 2013-06-02 Comment[AEExperimentType] ChIP-chip by tiling array Comment[AdditionalFile:Data1] GSE43384_130411_Rec114_t4_all_profiles_bw1000_decile_normalized.xls Comment[AdditionalFile:Data2] GSE43384_230811_Spo11_rad50S_-FA_t6_all_profiles_bw500_decile_normalized.xls Comment[AdditionalFile:Data3] GSE43384_WCE_4h_meiosis.CEL SDRF File E-GEOD-43384.sdrf.txt