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SUMO chains fine-tune DNA replication of repetitive sequences by restricting the access of recombination factors

ABSTRACT: SUMOylation, a protein post-translational modification present in all eukaryotes, involves the covalent attachment of SUMO (small ubiquitin-like modifier) to target proteins, modulating their function, localization or stability. One of the least understood features of SUMOylation is formation of polymeric chains and their impact on DNA metabolism. Here, using Schizosaccharomyces pombe,we demonstrated that cells devoid of SUMO chains exhibit elevated spontaneous replication stress and DNA damage. We found that SUMO chains promoted rDNA stability,controlled proper centromeric organization, and in their absence recombination factors accessed more frequently both of those loci. We directly showed increased fork stalling at rDNA and measured higher recombination rates at centromeres upon SUMO chains deficiency. Moreover, we successfully established the split-SUMO-ID proteomics approach in fission yeast to identify SUMO- and Rad52-dependent interactome. Our results suggest that SUMO chains maintain the stability of difficult-to-replicate loci by limiting the access of recombination factors to stalled replication forks.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE276805 | GEO | 2024/09/15

REPOSITORIES: GEO

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Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip â¢ The goal of the experiment Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae â¢ Experimental factor Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter) Cell condition 1: G1 arrest with alpha-factor Cell condition 2: HU treatment â¢ Experimental design ChIP analyses: ChIP using anti-c-Myc antibody. ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used. â¢ Quality control steps taken Confirmation of several loci by quantitative real time PCR. Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.

Project description:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Î mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells. Genome-wide distributions of Rad52 in wild type and in swi1 deletion in fission yeast The'SP1173_WT ChIP-seq' is an input sample (non-tagged data).

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SUMO chains fine-tune DNA replication of repetitive sequences by restricting the access of recombination factors

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