Unknown,Transcriptomics,Genomics,Proteomics

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Smc5/6-Mms21 prevents and eliminates inappropriate recombination intermediates in meiosis

ABSTRACT: Repairing broken chromosomes via joint molecule (JM) intermediates is hazardous and therefore strictly controlled in most organisms. Also in budding yeast meiosis, where production of enough crossovers via JMs is imperative, only a subset of DNA breaks are repaired via JMs, closely regulated by the ZMM pathway. The other breaks are repaired to non-crossovers avoiding JM formation requiring BLM/Sgs1 helicase. "Rogue" JMs that escaped the ZMM pathway and BLM/Sgs1 are eliminated before metaphase by resolvases like Mus81-Mms4 to prevent chromosome nondisjunction. 7 genome-wide meiotic ChIP-seq sets: 2 meiotic time points Smc6-myc DNA interaction (Smc6-ChIP), 2 meiotic time points Smc6-myc DNA interaction in the absence of induced recombination (Smc6-ChIP, spo11delta), 2 meiotic time points Sgs1-myc DNA interaction (Sgs1-ChIP), and 1 meiotic time point untagged as a negative control. (Corresponds to the main Figures 2 and 6, as well as to Figures S4, S7, in the associated publication.)

ORGANISM(S): Saccharomyces cerevisiae SK1

SUBMITTER: Franz Klein

PROVIDER: E-GEOD-51977 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA. Cell culture conditions and media Human fibroblast cell strains (WS: AG05229, AG12795, AG12797; BS: GM02932, GM03402, GM16891; RTS: AG18371, AG18375, AG05013; Normal/Wild-type: AG04054, AG06310, AG09975) were obtained from the Coriell Repository (Camden, NJ), from donors matched for gender and of similar ages, and were at similar passage levels. Cells were cultured in MEM supplemented with Earle’s salts, 20% fetal bovine serum, 1x penicillin/streptomycin, and 1x fungizone in 3% O2 at 37oC and harvested for RNA extraction during active growth and at ~ 80% confluence. GeneChip microarray expression Total RNA from the 12 fibroblast cell strains was isolated by extraction with TRIzol (Invitrogen) and purified using the RNeasy system (Qiagen). Total RNA was amplified by in vitro transcription using the Ovation RNA Amplification System V2 (NuGen). The resultant cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen), and then purified using QIAquick columns (Qiagen), as specified by the Ovation System manual. Labeled probe was hybridized to Affymetrix U133A 2.0 GeneChips, and ultimately scanned using an Axon GenePix array scanner. Statistical analysis of microarray expression experiment The output files were normalized by Robust Multiarray Average (RMA), using the R package GCRMA and gene expression levels were log2-transformed.

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Smc5/6-Mms21 prevents and eliminates inappropriate recombination intermediates in meiosis

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