ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.

The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily.

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3' and 5' flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5' ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.

DNA repair and global sumoylation are regulated by distinct Ubc9 noncovalent complexes.

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways.

ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair.

The Rad50 ABC-ATPase complex with Mre11 nuclease is essential for dsDNA break repair, telomere maintenance and ataxia telangiectasia-mutated kinase checkpoint signaling. How Rad50 affects Mre11 functions and how ABC-ATPases communicate nucleotide binding and ligand states across long distances and among protein partners are questions that have remained obscure. Here, structures of Mre11-Rad50 complexes define the Mre11 2-helix Rad50 binding domain (RBD) that forms a four-helix interface with Rad50 coiled coils adjoining the ATPase core. Newly identified effector and basic-switch helix motifs extend the ABC-ATPase signature motif to link ATP-driven Rad50 movements to coiled coils binding Mre11, implying an ~30-Å pull on the linker to the nuclease domain. Both RBD and basic-switch mutations cause clastogen sensitivity. Our new results characterize flexible ATP-dependent Mre11 regulation, defects in cancer-linked RBD mutations, conserved superfamily basic switches and motifs effecting ATP-driven conformational change, and they provide a unified comprehension of ABC-ATPase activities.

gammaH2A binds Brc1 to maintain genome integrity during S-phase.

ATM(Tel1) and ATR(Rad3) checkpoint kinases phosphorylate the C-terminus of histone H2AX (H2A in yeasts) in chromatin flanking DNA damage, establishing a recruitment platform for checkpoint and repair proteins. Phospho-H2A/X (gammaH2A/X)-binding proteins at double-strand breaks (DSBs) have been characterized, but those required for replication stress responses are unknown. Here, we present genetic, biochemical, small angle X-ray scattering (SAXS), and X-ray structural studies of the Schizosaccharomyces pombe Brc1, a 6-BRCT-domain protein that is structurally related to Saccharomyces cerevisiae Rtt107 and mammalian PTIP. Brc1 binds gammaH2A to form spontaneous and DNA damage-induced nuclear foci. Spontaneous Brc1 foci colocalize with ribosomal DNA repeats, a region prone to fork pausing and genomic instability, whereas DNA damage-induced Brc1 foci colocalize with DSB response factors. gammaH2A binding is critical for Brc1 function. The 1.45 A resolution crystal structure of Brc1-gammaH2A complex shows how variable BRCT insertion loops sculpt tandem-BRCT phosphoprotein-binding pockets to facilitate unique phosphoprotein-interaction specificities, and unveils an acidic DNA-mimicking Brc1 surface. From these results, Brc1 docking to gammaH2A emerges as a critical chromatin-specific response to replication-associated DNA damage.

Nbs1 flexibly tethers Ctp1 and Mre11-Rad50 to coordinate DNA double-strand break processing and repair.

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-BRCT(1)-BRCT(2) 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 bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of 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.

Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair.

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 define 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 fission 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).