Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells

Saleh‐Gohari, Nasrollah; Helleday, Thomas

doi:10.1038/sj.onc.1208178

Cited by 16 publications

(11 citation statements)

References 27 publications

(43 reference statements)

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“…We confirmed that an I-SceI-induced DSB produces mainly short-tract gene conversion products (Fig. 3B) (16,35). This is consistent with current models for homologous recombination triggered by DSBs, resulting in gene conversion (see Fig.…”

Section: Resultssupporting

confidence: 77%

Spontaneous Homologous Recombination Is Induced by Collapsed Replication Forks That Are Caused by Endogenous DNA Single-Strand Breaks

Saleh‐Gohari

Bryant

Schultz

et al. 2005

Molecular and Cellular Biology

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Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous ␥H2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that ␥H2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.

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Section: Resultssupporting

confidence: 77%

Spontaneous Homologous Recombination Is Induced by Collapsed Replication Forks That Are Caused by Endogenous DNA Single-Strand Breaks

Saleh‐Gohari

Bryant

Schultz

et al. 2005

Molecular and Cellular Biology

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281

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“…HR was investigated in four independently isolated CL-V4B clones following transient transfection with the pCMV3xnlsI-SceI vector, encoding the I-SceI restriction endonuclease, to make a DSB. Recombinant clones were selected with G418 and we found that the HR frequencies are overall lower in the RAD51C deficient cells as compared with V79 wild type control from earlier experiments ( Table 2 ) (Saleh-Gohari and Helleday, 2004). Also, the pooled data showed that HR in RAD51C deficient CL-V4B cells is significantly reduced as compared with HR in the corresponding wild type cells ( Fig.…”

Section: Rad51c Is Involved In Homologous Recombinationmentioning

confidence: 82%

RAD51C (RAD51L2) is involved in maintaining centrosome number in mitosis

Lindh

Schultz²,

Saleh‐Gohari³

et al. 2007

Cytogenet Genome Res

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The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes either XRCC2 or XRCC3. Both of these complexes may have important functions in homologous recombination (HR). Here, we confirm that the frequency of DNA double-strand break (DSB)-induced HR is reduced in the RAD51C deficient cell line CL-V4B, in agreement with a role for RAD51C in HR. We report that mitotic RAD51C deficient CL-V4B cells also have an increased number of centrosomes in mitosis resulting in aberrant mitotic spindles. These data suggest that the RAD51C protein is important in maintaining correct centrosome numbers and that the complexes including RAD51C and XRCC2 or XRCC3 may be of importance in maintaining correct centrosome numbers in mitosis. Increased centrosome numbers following a RAD51C defect indicates that this protein might be important in preventing aneuploidy, suggesting that it could be a potential tumour suppressor in mammals.

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“…This model explains the observed B-cell development defect in Xrcc2 Ϫ/Ϫ cells, as well as the early S-phase cell cycle arrest, and is supported by several additional lines of evidence. First, there is accumulating data that HR factors are required for replication fork restart following stalling or regression (28)(29)(30). Second, Xrcc2 is required, at least in some cells, for resistance to hydroxyureainduced replication stress (14).…”

Section: Discussionmentioning

confidence: 99%

Homologous Recombination Is Necessary for Normal Lymphocyte Development

Caddle

Hasham

Schott

et al. 2008

Molecular and Cellular Biology

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Primary immunodeficiencies are rare but serious diseases with diverse genetic causes. Accumulating evidence suggests that defects in DNA double-strand break (DSB) repair can underlie many of these syndromes. In this context, the nonhomologous end joining pathway of DSB repair is absolutely required for lymphoid development, but possible roles for the homologous recombination (HR) pathway have remained more controversial. While recent evidence suggests that HR may indeed be important to suppress lymphoid transformation, the specific relationship of HR to normal lymphocyte development remains unclear. We have investigated roles of the X-ray cross-complementing 2 (Xrcc2) HR gene in lymphocyte development. We show that HR is critical for normal B-cell development, with Xrcc2 nullizygosity leading to p53-dependent early S-phase arrest. In the absence of p53 (encoded by Trp53), Xrcc2-null B cells can fully develop but show high rates of chromosome and chromatid fragmentation. We present a molecular model wherein Xrcc2 is important to preserve or restore replication forks during rapid clonal expansion of developing lymphocytes. Our findings demonstrate a key role for HR in lymphoid development and suggest that Xrcc2 defects could underlie some human primary immunodeficiencies.Human immunodeficiencies are complex, multivariate diseases, the underlying genetic causes of which remain mostly unknown (9,11,31,32,35). In the past decade, defects in DNA double-strand break (DSB) repair have been implicated in some lymphodeficiencies, such as radiosensitive severe combined immunodeficiency. To resist the deleterious effects of DSBs, cells must efficiently respond to, and repair, the damage and maintain genomic integrity. Most eukaryotes employ two predominant DSB repair pathways: nonhomologous end joining (NHEJ), which catalyzes the religation of cognate broken DNA ends irrespective of sequence homology; and homologous recombination (HR), which utilizes a homologous DNA template to effect error-free DSB repair. While all cells likely incur random DSBs as a result of endogenous metabolism or exposure to environmental agents, certain cell types also deliberately generate DSBs to effect specific developmental programs (for review, see references 18 and 20). In this context, NHEJ is required to resolve DNA breaks associated with V(D)J recombination in developing lymphocytes, failure of which can lead to lymphodeficiency (2,20). In contrast, possible roles for the HR pathway, in either normal lymphocyte development or in lymphodeficiency, remain controversial. Recently the controversy has been rekindled by evidence that HR may be important to suppress T-cell lymphomagenesis (21).Here we investigate the role of the X-ray cross-complementing 2 (Xrcc2) gene in B-cell development. Xrcc2 is a member of the Rad51 gene family and was initially identified by its ability to complement DNA damage sensitivity in mutant Chinese hamster ovary (CHO) cells (34). Subsequent studies have shown XRCC2 to participate in one or more unique, multiprotei...

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Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells

Cited by 16 publications

References 27 publications

Spontaneous Homologous Recombination Is Induced by Collapsed Replication Forks That Are Caused by Endogenous DNA Single-Strand Breaks

Spontaneous Homologous Recombination Is Induced by Collapsed Replication Forks That Are Caused by Endogenous DNA Single-Strand Breaks

RAD51C (RAD51L2) is involved in maintaining centrosome number in mitosis

Homologous Recombination Is Necessary for Normal Lymphocyte Development

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