Dataset Information

HSSB1 DNA-binding is modulated by dynamic DNA-PK-mediated phosphorylation

ABSTRACT: The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the later process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and proper DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 may also be directly phosphorylated by DNA-PK at serine residue 134. While this modification does occur in undamaged cells, it is largely suppressed by PPP-family protein phosphatases. Following the disruption of replication forks, S134 phosphorylation is however enhanced and promotes cellular survival. Interestingly, while hSSB1 is known to associate with replication forks following their disruption, we here find that S134 phosphorylation may limit binding to replication fork junctions. Together, these data represent a novel mechanism for hSSB1 regulation following the inhibition of replication.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

SUBMITTER: Catherine Botting

LAB HEAD: Catherine Helen Botting

PROVIDER: PXD003964 | Pride | 2018-02-07

REPOSITORIES: Pride

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hSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases.

Ashton Nicholas W NW Ashton Nicholas W NW Paquet Nicolas N Shirran Sally L SL Bolderson Emma E Kariawasam Ruvini R Touma Christine C Fallahbaghery Azadeh A Gamsjaeger Roland R Cubeddu Liza L Botting Catherine C Pollock Pamela M PM O'Byrne Kenneth J KJ Richard Derek J DJ

DNA repair 20170406

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 i ...[more]

PMID: 28448822

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Project description:DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called “roadblocks” (e.g., DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.

Project description:The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

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	2YHU1Atryp5ul.wiff.zip	Wiff
	2YHU1Atryp5ul.wiff2.zip	Wiff
	2YHU1Atryp5ul.zip	Other
	F005785.mzid	Mzid