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 Mre11's 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.

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 doublestrand 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.double-strand break repair ͉ genome rearrangements D NA double-strand breaks (DSBs) are harmful lesions generated by a variety of endogenous or exogenous stresses, potentially leading to genomic rearrangement. Nonhomologous endjoining (NHEJ) is a prominent pathway for DSB repair (1). Canonical NHEJ involves the successive intervention of the KU80-KU70 heterodimer, DNA-PKcs-Artemis, and, finally, ligase IV (Lig4) associated with its cofactors XRCC4 and Cernunnos/Xlf (2, 3). KU-independent NHEJ (KU-alt) has been described in vitro in both acellular extracts and cultured cells (1, 4-6).Alternative, XRCC4-independent DSB repair pathways (XRCC4-alt) have also been described, using episomic plasmid in cultured cells, using pulse field gel electrophoresis, or in in vitro biochemical experiments (5-10). One hypothesis could propose that XRCC4 and KU are implicated in the same canonical NHEJ pathway, whereas the alternate pathway is independent of both KU and XRCC4. However, in transgenic mice, the inactivation of XRCC4 or Lig4 results in a more severe phenotype than the inactivation of KU (11), suggesting that XRCC4 might have an additional essential function; however, studies show that XRCC4 and Lig4 do not have roles outside of NHEJ, whereas in contrast, KU acts in other processes such as transcription, apoptosis, and responses to the cell microenvironment (12)(13)(14).Alternatively, these varying phenotypes in mice may actually result from differences in DSB repair efficiencies, indicating that defects in XRCC4 might be more deleterious for DSB repair than defects in KU. What challenges this hypothesis, however, is that substantial class switch recombination (CSR) has recently been shown to occur in mouse B cells without XRCC4, whereas no CSR was recorded in cells devoid of KU (15-18).The relative contributions of XRCC4 and KU80 versus the XRCC4-alt and KU-alt pathways, respectively, to DSB repair remain unclear in wild-type cells. Contrasting results were obtained in living cells, using an episomic plasm...

The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp) 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.