Dataset Information

Stn1 S74 phosphorylation - Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions by conventional DNA polymerases and telomerase, though the regulation of this process is not fully understood

ABSTRACT: Publication abstract: Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions by conventional DNA polymerases and telomerase, though the regulation of this process is not fully understood. Telomere replication requires (C)-Stn1-Ten1, a telomere ssDNA-binding complex that is homologous to RPA. Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at S74, a residue that lies within the conserved OB fold domain. Consequently, ssu72Δ mutants are defective in telomere replication and exhibit long 3′ overhangs, which are indicative of defective lagging strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling the recruitment of hSTN1 to telomeres. Thus, in this study, we demonstrate a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging strand DNA synthesis, thus terminating the cycle of telomere replication. Summary of MS experiment: Stn1 protein was tagged in the C-terminus with 13-myc tag and purified by immunoprecipitation. The purified extracts were separated by SDS-PAGE and proteins between 63 and 75 kDa were excised and in-gel digested. Tryptic peptides were analyzed by LC-MS/MS using an AB Sciex TripleTOF 6600 mass spectrometer upon separation by nanoLC-MS using an Ekspert 425 nanoLC with cHiPLC. Stn1 protein was identified with 48% of coverage and Serine-74 was found to be phosphorylated.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Schizosaccharomyces Pombe 927

SUBMITTER: Inês M. Luís

LAB HEAD: Isabel A. Abreu

PROVIDER: PXD011953 | Pride | 2019-02-19

REPOSITORIES: Pride

ACCESS DATA

Similar Datasets

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).

Dataset Information

Stn1 S74 phosphorylation - Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions by conventional DNA polymerases and telomerase, though the regulation of this process is not fully understood

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure