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The conserved Mre11-Rad50 complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks. While it is known that Mre11-Rad50 foci formation at DNA lesions accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae Mre11-Rad50 with or without Xrs2 forms higher-order assemblies in solution and on DNA. Rad50 mediates such oligomerization, and mutations in a conserved Rad50 beta-sheet enhance or disrupt oligomerization. We demonstrate that Mre11-Rad50-Xrs2 oligomerization facilitates foci formation, DNA damage signaling, repair, and telomere maintenance in vivo. Mre11-Rad50 oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5′-DNA strand near double-strand breaks. Interestingly, mutations in the human RAD50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide insights into their potential role in chemoresistance. The Mre11-Rad50 (MR) complex has key functions in the detection, signaling and repair of DNA breaks. Here the authors use transmission electron microscopy to show MR oligomerization is governed by a small beta-sheet protruding from the head domain of Rad50 at the base of the MR structure, and reveal MR head domain oligomerization is required for efficient DNA end resection.

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2. An enzymatic ensemble including Dna2 functions in DNA end resection; the function of the single-stranded DNA binding protein RPA in this complex has been underappreciated. Here the authors employ molecular modeling, biochemistry, and single molecule biophysics to reveal RPA directly promotes Dna2 recruitment, nuclease and helicase activities.

The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein’s in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery. The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes. Here the authors show that ComFC is a membrane-associated protein that participates in the transport of DNA through the cell membrane and the handling of the single-stranded DNA once delivered into the cytoplasm.

HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA. MCM8-9 and HROB function together in DNA damage response. Here, the authors describe the mechanism of DNA unwinding by MCM8-9 and its activation by HROB. HROB makes direct contacts with both MCM8 and MCM9 and promotes DNA unwinding downstream of MCM8-9 loading and hexameric ring formation on DNA.

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between theMutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. PPP2R1A is found to associate at the lamellipodial edge with an alternative form of WAVE complex, the WAVE Shell Complex, that contains NHSL1 instead of the Arp2/3 activating subunit WAVE, as in the canonical WAVE Regulatory Complex. PPP2R1A is required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement is abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impair WAVE Shell Complex binding and migration regulation, suggesting that the coupling of PPP2R1A to the WAVE Shell Complex is essential to its function. The WAVE regulatory complex activates Arp2/3 at the cell cortex and in membrane protrusions to generate persistent cell migration. Here authors show that PPP2R1A, a scaffold subunit of protein phosphatase 2, associates with an alternative form of the WAVE complex where WAVE, the subunit that activates Arp2/3, is replaced by NHSL1.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5′-terminated strands at DSB sites 1 , 2 . The breast cancer suppressor BRCA1–BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress 1 , 3 , 4 , 5 , 6 , 7 , 8 – 9 . BRCA1–BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear 10 , 11 , 12 , 13 , 14 , 15 – 16 . Using purified recombinant proteins, we show here that BRCA1–BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1–BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11–RAD50–NBS1 and phosphorylated CtIP, BRCA1–BARD1 forms the BRCA1–C complex 17 , 18 , which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1–C complex 19 , 20 – 21 , inhibits resection, showing that BRCA1–C is a functionally integrated ensemble. Whereas BRCA1–BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4 , 5 – 6 , 8 ). We show that in the presence of RAD51, BRCA1–BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1–BARD1 in various physiological contexts. BRCA1–BARD1 directly promotes double-strand break repair by stimulating long-range DNA end resection pathways.

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLβ complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

Histone H3K4 methylation is a feature of meiotic recombination hotspots shared by many organisms including plants and mammals. Meiotic recombination is initiated by programmed double-strand break (DSB) formation that in budding yeast takes place in gene promoters and is promoted by histone H3K4 di/trimethylation. This histone modification is recognized by Spp1, a PHD finger containing protein that belongs to the conserved histone H3K4 methyltransferase Set1 complex. During meiosis, Spp1 binds H3K4me3 and interacts with a DSB protein, Mer2, to promote DSB formation close to gene promoters. How Set1 complex- and Mer2- related functions of Spp1 are connected is not clear. Here, combining genome-wide localization analyses, biochemical approaches and the use of separation of function mutants, we show that Spp1 is present within two distinct complexes in meiotic cells, the Set1 and the Mer2 complexes. Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation. Conversely, the Spp1-Mer2 interaction is required for normal meiotic recombination initiation, but dispensable for Set1 complex-mediated histone H3K4 methylation. Finally, we provide evidence that Spp1 preserves normal H3K4me3 levels independently of the Set1 complex. We propose a model where Spp1 works in three ways to promote recombination initiation: first by depositing histone H3K4 methylation (Set1 complex), next by \"reading\" and protecting histone H3K4 methylation, and finally by making the link with the chromosome axis (Mer2-Spp1 complex). This work deciphers the precise roles of Spp1 in meiotic recombination and opens perspectives to study its functions in other organisms where H3K4me3 is also present at recombination hotspots.

Heterodimeric complexes containing the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) are regarded as central regulators of plant innate immunity. In this context, a complex of EDS1 with PHYTOALEXIN DEFICIENT4 (PAD4) is required for basal resistance and signaling downstream of immune receptors containing an N-terminal Toll-interleukin-1 receptor-like domain (TNLs) in Arabidopsis (Arabidopsis thaliana). Here we analyze EDS1 functions in the model Solanaceous plant Nicotiana benthamiana (Nb). Stable Nb mutants deficient in EDS1 complexes are not impaired in basal resistance, a finding which contradicts a general role for EDS1 in immunity. In Nb, PAD4 demonstrated no detectable immune functions, but TNL-mediated resistance responses required EDS1 complexes incorporating a SENESCENCE ASSOCIATED GENE101 (SAG101) isoform. Intriguingly, SAG101 is restricted to those genomes also encoding TNL receptors, and we propose it may be required for TNL-mediated immune signaling in most plants, except the Brassicaceae. Transient complementation in Nb was used for accelerated mutational analyses while avoiding complex biotic interactions. We identify a large surface essential for EDS1-SAG101 immune functions that extends from the N-terminal lipase domains to the C-terminal EDS1-PAD4 domains and might mediate interaction partner recruitment. Furthermore, this work demonstrates the value of genetic resources in Nb, which will facilitate elucidation of EDS1 functions.