Dataset Information

Roles of XRCC2, RAD51B and RAD51D in RAD51-independent SSA recombination.

ABSTRACT: The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.

SUBMITTER: Serra H

PROVIDER: S-EPMC3836719 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Roles of XRCC2, RAD51B and RAD51D in RAD51-independent SSA recombination.

Serra Heïdi H Da Ines Olivier O Degroote Fabienne F Gallego Maria E ME White Charles I CI

PLoS genetics 20131121 11

The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by ...[more]

PMID: 24278037

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Project description:PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. METHODS:Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and ?H2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. RESULTS:Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. CONCLUSIONS:Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

Dataset Information

Roles of XRCC2, RAD51B and RAD51D in RAD51-independent SSA recombination.

Publications

Roles of XRCC2, RAD51B and RAD51D in RAD51-independent SSA recombination.

Similar Datasets

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