SUMMARY Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To de?ne the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of ?ssion yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).

It has been reported that soluble interleukin (IL)-6 receptor (sIL-6R) is detected in the serum of healthy individuals and its level is increased in patients with multiple myeloma and human immunodeficiency virus infection. Although several reports have suggested that sIL-6R potentiates 1L-6 action, its physiological role remains unclear. In this study, we examined the role of sIL-6R on osteoclast formation by IL-6, using a coculture of mouse osteoblasts and bone marrow cells. Neither recombinant mouse IL-6 (mIL-6) nor mouse sIL-6R (smIL-6R) induced osteoclast-like multinucleated cell (MNC) formation when they were added separately. In contrast, simultaneous treatment with mIL-6 and smIL-6R strikingly induced MNC formation. These MNCs satisfied major criteria of authentic osteoclasts, such as tartrateresistant acid phosphatase (TRAP) activity, calcitonin receptors, and pit formation on dentine slices. The MNC formation induced by mIL-6 and smIL-6R was dose-dependently inhibited by adding monoclonal anti-mouse IL-6R antibody (MR16-1). It is likely that osteoblasts and osteoclast progenitors are capable of transducing a signal from a complex of IL-6 and sIL-6R through gpl3O, even though they may have no or a very small number of IL-6Rs. Factors such as IL-1l, oncostatin M, and leukemia inhibitory factor, which are known to exert their functions through gpl3O (the signal-transducing chain of IL-6R), also induced MNC formation in our coculture system. These results suggest that increased circulating or locally produced sIL-6R induces osteoclast formation in the presence of IL-6 mediated by a mechanism involving gpl3O. This may play an important physiological or pathological role in conditions associated with increased osteoclastic bone resorption.

The Mre11-Rad50-Nbs1 (MRN) complex is a primary sensor of DNA double-strand breaks (DSBs). Upon recruitment to DSBs, it plays a critical role in catalyzing 5' --> 3' single-strand resection that is required for repair by homologous recombination (HR). Unknown mechanisms repress HR in G1 phase of the cell cycle during which nonhomologous end-joining (NHEJ) is the favored mode of DSB repair. Here we describe fission yeast Ctp1, so-named because it shares conserved domains with the mammalian tumor suppressor CtIP. Ctp1 is recruited to DSBs where it is essential for repair by HR. Ctp1 is required for efficient formation of RPA-coated single-strand DNA adjacent to DSBs, indicating that it functions with the MRN complex in 5' --> 3' resection. Transcription of ctp1(+) is periodic during the cell cycle, with the onset of its expression coinciding with the start of DNA replication. These data suggest that regulation of Ctp1 underlies cell-cycle control of HR.

SUMMARY The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a divalent MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2 0 -deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as earlyfiring origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.