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Replication fork stability is essential for the maintenance of centromere integrity in the absence of heterochromatin.

ABSTRACT: The centromere of many eukaryotes contains highly repetitive sequences marked by methylation of histone H3K9 by Clr4(KMT1). This recruits multiple heterochromatin proteins, including Swi6 and Chp1, to form a rigid centromere and ensure accurate chromosome segregation. In the absence of heterochromatin, cells show an increased rate of recombination in the centromere, as well as chromosome loss. These defects are severely aggravated by loss of replication fork stability. Thus, heterochromatin proteins and replication fork protection mechanisms work in concert to prevent abnormal recombination, preserve centromere integrity, and ensure faithful chromosome segregation.

SUBMITTER: Li PC

PROVIDER: S-EPMC3652564 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Replication fork stability is essential for the maintenance of centromere integrity in the absence of heterochromatin.

Li Pao-Chen PC Petreaca Ruben C RC Jensen Amanda A Yuan Ji-Ping JP Green Marc D MD Forsburg Susan L SL

Cell reports 20130307 3

The centromere of many eukaryotes contains highly repetitive sequences marked by methylation of histone H3K9 by Clr4(KMT1). This recruits multiple heterochromatin proteins, including Swi6 and Chp1, to form a rigid centromere and ensure accurate chromosome segregation. In the absence of heterochromatin, cells show an increased rate of recombination in the centromere, as well as chromosome loss. These defects are severely aggravated by loss of replication fork stability. Thus, heterochromatin prot ...[more]

PMID: 23478021

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Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and Î³H2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis S. cerevisiae chromosomes IIIâV, and chromosome VI highdensity oligonucleotide microarrays were provided by Affymetrix Custom Express Service (SC3456a520015F, P/N 520015; rikDACF, P/N 510636, respectively). Sequence and position of oligonucleotides on the microarrays are available from Affymetrix. ChIP was carried out as previously described (Katou et al. 2003; Katou et al. 2006): we disrupted 1.5 x 108 cells byMulti-beads shocker (MB400U, Yasui Kikai) using glass beads. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc.) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich) were used for chromatin immunoprecipitation. ChIPed DNA was purified and amplified by random priming as described (Katou et al. 2003): a total of 10 Î¼g of amplified DNA was digested with DNaseI to a mean size of 100 bp, purified, and the fragments were end-labelled with biotin-N6-ddATP23. Hybridization, washing, staining and scanning were performed according to the manufacturerâs instruction (Affymetrix). Primary data analyses were carried out using the Affymetrix microarray Suite version 5.0 software to obtain hybridization intensity, fold change value, change P-value and detection P-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIPed fraction with supernatant fraction by using three criteria. First, the reliability of strength of signal was judged by detection P-value of each locus (P â¥0.025). Second, reliability of binding ratio was judged by change P-value (P â¥ 0.025). Third, clusters consisting of at least three contiguous loci that filled the above Bermejo et al. 26 two criteria were selected, because it was known that a single site of proteinâDNA interaction will result in immunoprecipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbours. For the analyses of BrdU incorporation, cells were fixed by ice-cold buffer containing 0.1% azide, and then total DNA from 3 x108 cells was purified. DNA was sheared to 300 bp by sonication, denatured, and mixed with 2Î¼g anti-BrdU monoclonal antibody (2B1D5F5H4E2; MBL). Antibody-bound and unbound fractions were subsequently purified, amplified, labelled and hybridized to the DNA chip.

Dataset Information

Replication fork stability is essential for the maintenance of centromere integrity in the absence of heterochromatin.

Publications

Replication fork stability is essential for the maintenance of centromere integrity in the absence of heterochromatin.

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