Nitrogen and phosphorous acquisition strategies drive coexistence patterns among archaeal lineages in soil.

Soil represents the largest reservoir of Archaea on Earth. Present-day archaeal diversity in soils globally is dominated by members of the class Nitrososphaeria. The evolutionary radiation of this class is thought to reflect adaptations to a wide range of temperatures, pH, and other environmental conditions. However, the mechanisms that govern competition and coexistence among Nitrososphaeria lineages in soil remain poorly understood. Here we show that predominant soil Nitrososphaeria lineages compose a patchwork of gene inventory and expression profiles for ammonia, urea, and phosphate utilization. In contrast, carbon fixation, respiration, and ATP synthesis genes are conserved and expressed consistently among predominant phylotypes across 12 major evolutionary lineages commonly found in soil. In situ gene expression profiles closely resemble pure culture reference strains under optimal growth conditions. Together, these results reveal resource-based coexistence patterns among Nitrososphaeria lineages and suggest complementary ecophysiological niches associated with differential nutrient acquisition strategies among globally predominant archaeal lineages in soil.

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes.

It has been predicted that 30 to 80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing an ∼5-fold average coverage of the T. kodakarensis genome were prepared in Escherichia coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') that is found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing, and capillary electrophoresis. The method described here provides the details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next-generation sequencing, and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities, including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

Chromosome segregation in Archaea: SegA- and SegB-DNA complex structures provide insights into segrosome assembly.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea.

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.

The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase.

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase.

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5'-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.

Comparative genomics of DNA-binding transcription factors in archaeal and bacterial organisms.

Archaea represent a diverse phylogenetic group that includes free-living, extremophile, mesophile, symbiont, and opportunistic organisms. These prokaryotic organisms share a high significant similarity with the basal transcriptional machinery of Eukarya, and they share regulatory mechanisms with Bacteria, such as operonic organization and DNA-binding transcription factors (TFs). In this work, we identified the repertoire of TFs in 415 archaeal genomes and compared them with their counterparts in bacterial genomes. The comparisons of TFs, at a global level and per family, allowed us to identify similarities and differences between the repertoires of regulatory proteins of bacteria and archaea. For example, 11 of 62 families are more highly abundant in archaea than bacteria, and 13 families are abundant in bacteria but not in archaea and 38 families have similar abundances in the two groups. In addition, we found that archaeal TFs have a lower isoelectric point than bacterial proteins, i.e., they contain more acidic amino acids, and are smaller than bacterial TFs. Our findings suggest a divergence occurred for the regulatory proteins, even though they are common to archaea and bacteria. We consider that this analysis contributes to the comprehension of the structure and functionality of regulatory proteins of archaeal organisms.

Functional compartmentalization and metabolic separation in a prokaryotic cell.

The prokaryotic cell is traditionally seen as a "bag of enzymes," yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the "cytoplasm" or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Single-Stranded DNA-Binding Proteins in the Archaea.

Single-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain architectures and subunit compositions, with OB-fold ssDNA-binding domains being a characteristic of most, but not all. This chapter summarizes current knowledge of the distribution, structure, and biological function of the archaeal ssDNA-binding factors, highlighting key features shared between clades and those that distinguish the proteins of different clades from one another. The likely cellular functions of the proteins are discussed and gaps in current knowledge identified.

A rod conformation of the Pyrococcus furiosus Rad50 coiled coil.

The Rad50-Mre11 nuclease complex plays a vital role in DNA repair in all domains of life. It recognizes and processes DNA double-strand breaks. Rad50 proteins fold into an extended structure with a 20 to 60 nm long coiled coil connecting a globular ABC ATPase domain with a zinc hook dimerization domain. A published structure of an archaeal Rad50 zinc hook shows coiled coils pointing away from each other. Here we present the crystal structure of an alternate conformation displaying co-aligned coiled coils. Archaeal Rad50 may thus switch between rod-shaped and ring-like conformations as recently proposed for a bacterial homolog.

Archaea: The Final Frontier of Chromatin.

The three domains of life employ various strategies to organize their genomes. Archaea utilize features similar to those found in both eukaryotic and bacterial chromatin to organize their DNA. In this review, we discuss the current state of research regarding the structure-function relationships of several archaeal chromatin proteins (histones, Alba, Cren7, and Sul7d). We address individual structures as well as inferred models for higher-order chromatin formation. Each protein introduces a unique phenotype to chromatin organization, and these structures are put into the context of in vivo and in vitro data. We close by discussing the present gaps in knowledge that are preventing further studies of the organization of archaeal chromatin, on both the organismal and domain level.

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.

Modified base-binding EVE and DCD domains: striking diversity of genomic contexts in prokaryotes and predicted involvement in a variety of cellular processes.

BACKGROUND: DNA and RNA of all cellular life forms and many viruses contain an expansive repertoire of modified bases. The modified bases play diverse biological roles that include both regulation of transcription and translation, and protection against restriction endonucleases and antibiotics. Modified bases are often recognized by dedicated protein domains. However, the elaborate networks of interactions and processes mediated by modified bases are far from being completely understood. RESULTS: We present a comprehensive census and classification of EVE domains that belong to the PUA/ASCH domain superfamily and bind various modified bases in DNA and RNA. We employ the "guilt by association" approach to make functional inferences from comparative analysis of bacterial and archaeal genomes, based on the distribution and associations of EVE domains in (predicted) operons and functional networks of genes. Prokaryotes encode two classes of EVE domain proteins, slow-evolving and fast-evolving ones. Slow-evolving EVE domains in α-proteobacteria are embedded in conserved operons, potentially involved in coupling between translation and respiration, cytochrome c biogenesis in particular, via binding 5-methylcytosine in tRNAs. In ß- and γ-proteobacteria, the conserved associations implicate the EVE domains in the coordination of cell division, biofilm formation, and global transcriptional regulation by non-coding 6S small RNAs, which are potentially modified and bound by the EVE domains. In eukaryotes, the EVE domain-containing THYN1-like proteins have been reported to inhibit PCD and regulate the cell cycle, potentially, via binding 5-methylcytosine and its derivatives in DNA and/or RNA. We hypothesize that the link between PCD and cytochrome c was inherited from the α-proteobacterial and proto-mitochondrial endosymbiont and, unexpectedly, could involve modified base recognition by EVE domains. Fast-evolving EVE domains are typically embedded in defense contexts, including toxin-antitoxin modules and type IV restriction systems, suggesting roles in the recognition of modified bases in invading DNA molecules and targeting them for restriction. We additionally identified EVE-like prokaryotic Development and Cell Death (DCD) domains that are also implicated in defense functions including PCD. This function was inherited by eukaryotes, but in animals, the DCD proteins apparently were displaced by the extended Tudor family proteins, whose partnership with Piwi-related Argonautes became the centerpiece of the Piwi-interacting RNA (piRNA) system. CONCLUSIONS: Recognition of modified bases in DNA and RNA by EVE-like domains appears to be an important, but until now, under-appreciated, common denominator in a variety of processes including PCD, cell cycle control, antivirus immunity, stress response, and germline development in animals.

Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases.

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2' modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

Deciphering the functional diversity of DNA-binding transcription factors in Bacteria and Archaea organisms.

DNA-binding Transcription Factors (TFs) play a central role in regulation of gene expression in prokaryotic organisms, and similarities at the sequence level have been reported. These proteins are predicted with different abundances as a consequence of genome size, where small organisms contain a low proportion of TFs and large genomes contain a high proportion of TFs. In this work, we analyzed a collection of 668 experimentally validated TFs across 30 different species from diverse taxonomical classes, including Escherichia coli K-12, Bacillus subtilis 168, Corynebacterium glutamicum, and Streptomyces coelicolor, among others. This collection of TFs, together with 111 hidden Markov model profiles associated with DNA-binding TFs collected from diverse databases such as PFAM and DBD, was used to identify the repertoire of proteins putatively devoted to gene regulation in 1321 representative genomes of Archaea and Bacteria. The predicted regulatory proteins were posteriorly analyzed in terms of their genomic context, allowing the prediction of functions for TFs and their neighbor genes, such as genes involved in virulence, enzymatic functions, phosphorylation mechanisms, and antibiotic resistance. The functional analysis associated with PFAM groups showed diverse functional categories were significantly enriched in the collection of TFs and the proteins encoded by the neighbor genes, in particular, small-molecule binding and amino acid transmembrane transporter activities associated with the LysR family and proteins devoted to cellular aromatic compound metabolic processes or responses to drugs, stress, or abiotic stimuli in the MarR family. We consider that with the increasing data derived from new technologies, novel TFs can be identified and help improve the predictions for this class of proteins in complete genomes. The complete collection of experimentally characterized and predicted TFs is available at http://web.pcyt.unam.mx/EntrafDB/.

The N-terminal domain of Staphylothermus marinus McrB shares structural homology with PUA-like RNA binding proteins.

McrBC is a conserved modification-dependent restriction system that in Escherichia coli specifically targets foreign DNA containing methylated cytosines. Crystallographic data show that the N-terminal domain of Escherichia coli McrB binds substrates via a base flipping mechanism. This region is poorly conserved among the plethora of McrB homologs, suggesting that other species may use alternative binding strategies and/or recognize different targets. Here we present the crystal structure of the N-terminal domain from Stayphlothermus marinus McrB (Sm3-180) at 1.92 Å, which adopts a PUA-like EVE fold that is closely related to the YTH and ASCH RNA binding domains. Unlike most PUA-like domains, Sm3-180 binds DNA and can associate with different modified substrates. We find the canonical 'aromatic cage' binding pocket that confers specificity for methylated bases in other EVE/YTH domains is degenerate and occluded in Sm3-180, which may contribute to its promiscuity in target recognition. Further structural comparison between different PUA-like domains identifies motifs and conformational variations that correlate with the preference for binding either DNA or RNA. Together these data have important implications for PUA-like domain specificity and suggest a broader biological versatility for the McrBC family than previously described.

Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea.

Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show that both DNA polymerases, family-B polymerase (PolB) and family-D polymerase (PolD), can take charge of processing the RadA-mediated recombination intermediates. Our results also indicate that PolD is far less efficient, as compared with PolB, to extend the invaded DNA at the displacement-loop (D-loop) substrate. These observations coincide with previous genetic analyses obtained on Thermococcus species showing that PolB is mainly involved in DNA repair without being essential probably because PolD could take over combined with additional partners.

Mechanistic insights into Lhr helicase function in DNA repair.

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the 'parental' DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy.

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.