Pascale Bertrand

Role of Mre11 in chromosomal nonhomologous end joining in mammalian cells

Here we have used an intrachromosomal substrate to monitor the end joining of distant ends, which leads to DNA rearrangements in mammalian cells. We show that silencing Mre11 reduces the efficiency of nonhomologous end joining (NHEJ), affecting both the canonical and alternative pathways, partly in a manner that is independent of the ataxia-telangiectasia mutated kinase (ATM). Silencing of Rad50 or CtIP decreases end-joining efficiency in the same pathway as Mre11. In cells defective for Xrcc4, the MRE11–RAD50–NBS1 (MRN) complex inhibitor MIRIN decreases end-joining frequencies, demonstrating a role for MRN in alternative NHEJ. Consistently, MIRIN sensitizes both complemented and NHEJ-defective cells to ionizing radiation. Conversely, overexpression of Mre11 stimulates the resection of single-stranded DNA and increases alternative end joining, through a mechanism that requires Mre11s nuclease activity, but in an ATM-independent manner. These data demonstrate that, in addition to its role in ATM activation, Mre11 can favor alternative NHEJ through its nuclease activity.

Down‐regulation of BRCA1 expression by miR‐146a and miR‐146b‐5p in triple negative sporadic breast cancers

Germ‐line mutations in the BRCA1 gene strongly predispose women to breast cancer (lifetime risk up to 80%). Furthermore, the BRCA1 protein is absent or present at very low levels in about one third of sporadic breast cancers. However, the mechanisms underlying BRCA1 somatic inactivation appear multiple and are still not fully understood. We report here the involvement of miR‐146a and miR‐146b‐5p that bind to the same site in the 3′UTR of BRCA1 and down‐regulate its expression as demonstrated using reporter assays. This was further confirmed with the endogenous BRCA1 gene by transfecting microRNA (miRNA) precursors or inhibitors in mammary cell lines. This down‐regulation was accompanied by an increased proliferation and a reduced homologous recombination rate, two processes controlled by BRCA1. Furthermore, we showed that the highest levels of miR‐146a and/or miR‐146b‐5p are found in basal‐like mammary tumour epithelial cell lines and in triple negative breast tumours, which are the closest to tumours arising in carriers of BRCA1 mutations. This work provides further evidence for the involvement of miRNAs in sporadic breast cancer through down‐regulation of BRCA1.

Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein

Homologous recombination plays an essential role in processes involved in genome stability/instability, such as molecular evolution, gene diversification, meiotic chromosome segregation, DNA repair and chromosomal rearrangements. p53 devoid cells exhibit predisposition to neoplasia, defects in G1 checkpoint and high genetic instability but a normal rate of point mutations. We investigated the effect of a p53 mutation, on spontaneous homologous recombination between intrachromosomal direct repeat sequences, in mouse L cells. In these cells, wild type for the p53 gene, we have overexpressed the mutant p53175(Arg>His) protein leading to a p53 mutant phenotype, as verified by the absence of a G1 arrest after γ-irradiation. We show that the rate of spontaneous recombination is increased from five- to 20-fold in the mutant p53 lines. Moreover, this increase is observed in gene conversion as well as in deletion events. Our results provide new insights into the molecular mechanisms of genetic instability due to a defect of p53.

Is Non-Homologous End-Joining Really an Inherently Error-Prone Process?

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV–dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends.

XRCC4-null mice have a more severe phenotype than KU80-null mice. Here, we address whether this difference in phenotype is connected to nonhomologous end-joining (NHEJ). We used intrachromosomal substrates to monitor NHEJ of two distal double-strand breaks (DSBs) targeted by I-SceI, in living cells. In xrcc4-defective XR-1 cells, a residual but significant end-joining process exists, which primarily uses microhomologies distal from the DSB. However, NHEJ efficiency was strongly reduced in xrcc4-defective XR-1 cells versus complemented cells, contrasting with KU-deficient xrs6 cells, which showed levels of end-joining similar to those of complemented cells. Nevertheless, sequence analysis of the repair junctions indicated that the accuracy of end-joining was strongly affected in both xrcc4-deficient and KU-deficient cells. More specifically, these data showed that the KU80/XRCC4 pathway is conservative and not intrinsically error-prone but can accommodate non-fully complementary ends at the cost of limited mutagenesis.

Oxidative stress induces an ATM-independent senescence pathway through p38 MAPK-mediated lamin B1 accumulation

We report crosstalk between three senescence‐inducing conditions, DNA damage response (DDR) defects, oxidative stress (OS) and nuclear shape alterations. The recessive autosomal genetic disorder Ataxia telangiectasia (A‐T) is associated with DDR defects, endogenous OS and premature ageing. Here, we find frequent nuclear shape alterations in A‐T cells, as well as accumulation of the key nuclear architecture component lamin B1. Lamin B1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild‐type cells, and normalizing lamin B1 levels in A‐T cells reciprocally reduces both nuclear shape alterations and senescence. We further show that OS increases lamin B1 levels through p38 Mitogen Activated Protein kinase activation. Lamin B1 accumulation and nuclear shape alterations also occur during stress‐induced senescence and oncogene‐induced senescence (OIS), two canonical senescence situations. These data reveal lamin B1 as a general molecular mediator that controls OS‐induced senescence, independent of established Ataxia Telangiectasia Mutated (ATM) roles in OIS.

Human POMp75 is identified as the pro-oncoprotein TLS/FUS: both POMp75 and POMp100 DNA homologous pairing activities are associated to cell proliferation.

We have previously developed an assay to measure DNA homologous pairing activities in crude extracts: The POM blot. In mammalian nuclear extracts, we detected two major DNA homologous pairing activities: POMp100 and POMp75. Here, we present the purification and identification of POMp75 as the pro-oncoprotein TLS/FUS. Because of the pro-oncogene status of TLS/FUS, we studied in addition, the relationships between cell proliferation and POM activities. We show that transformation of human fibroblasts by SV40 large T antigen results in a strong increase of both POMp100 and TLS/POMp75 activities. Although detectable levels of both POMp100 and TLS/POMp75 are observed in non-immortalized fibroblasts or lymphocytes, fibroblasts at mid confluence or lymphocytes stimulated by phytohaemaglutinin, show higher levels of POM activities. Moreover, induction of differentiation of mouse F9 line by retinoic acid leads to the inhibition of both POMp100 and TLS/POMp75 activities. Comparison of POM activity of TLS/FUS with the amount of TLS protein detected by Western blot, suggests that the POM activity could be regulated by post-translation modification. Taken together, these results indicate that POMp100 and TLS/POMp75 activitites are present in normal cells but are connected to cell proliferation. Possible relationship between cell proliferation, response to DNA damage and DNA homologous pairing activity of the pro-oncoprotein TLS/FUS are discussed.

The DNA polymerase λ is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway.

Overexpression of mammalian Rad51 does not stimulate tumorigenesis while a dominant-negative Rad51 affects centrosome fragmentation, ploidy and stimulates tumorigenesis, in p53-defective CHO cells

Rad51 protein plays a pivotal role in homologous recombination (HR), which is involved in double-strand break repair and in genome maintenance. Despite interactions with tumor suppressor proteins, the role of mammalian Rad51 and more generally of HR in tumor prevention is not clearly established. Indeed, both high and low frequencies of HR as well as high and low levels of RAD51 expression have been reported in tumors and in precancerous conditions. To address the question of the impact of HR on tumorigenesis, we used Chinese hamster ovary (CHO) p53-defective cell lines overexpressing the mouse MmRAD51, which stimulates HR (we name these lines: Hyper-rec lines). In parallel, we used CHO cell lines expressing a RAD51 dominant-negative form that specifically inhibits gene conversion without affecting cell viability (Hypo-rec lines). These different lines were injected into nude mice to measure their tumorigenicity. Hypo-rec lines generated a higher frequency of tumors, which also exhibited faster growth, compared to control and Hyper-rec lines. Consistent with tumorigenicity, Hypo-rec cells exhibit spontaneous centrosome duplication defects and aneuploidy. These results are the first direct evidence of involvement of RAD51 in tumor repression.

A Role for BLM in Double-Strand Break Repair Pathway Choice: Prevention of CtIP/Mre11-Mediated Alternative Nonhomologous End-Joining

The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that thexa0Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200xa0bp) generated by alternative end-joining (A-EJ). BLM represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role for BLM that influences the DSB repair pathway choice: (1) protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2) promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid unscheduled resection that might jeopardize genome integrity.