Molecular architecture of the HerA–NurA DNA double-strand break resection complex

DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase–nuclease systems to generate 3′ single strand tails. In archaea, this requires the Mre11–Rad50 complex and the ATP-dependent helicase–nuclease complex HerA–NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA–NurA at 7.4 Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA’s nuclease active sites requires dsDNA to pass through a 23 Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.     

Structural and functional insights into DNA-end processing by the archaeal HerA helicase-NurA nuclease complex

Helicase-nuclease systems dedicated to DNA end resection in preparation for homologous recombination (HR) are present in all kingdoms of life. In thermophilic archaea, the HerA helicase and NurA nuclease cooperate with the highly conserved Mre11 and Rad50 proteins during HR-dependent DNA repair. Here we show that HerA and NurA must interact in a complex with specific subunit stoichiometry to process DNA ends efficiently. We determine crystallographically that NurA folds in a toroidal dimer of intertwined RNaseH-like domains. The central channel of the NurA dimer is too narrow for double-stranded DNA but appears well suited to accommodate one or two strands of an unwound duplex. We map a critical interface of the complex to an exposed hydrophobic epitope of NurA abutting the active site. Based upon the presented evidence, we propose alternative mechanisms of DNA end processing by the HerA-NurA complex.     

A bipolar DNA helicase gene, herA, clusters with rad50, mre11 and nurA genes in thermophilic archaea

We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon-like structure with a third gene (nurA) encoding a 5' to 3' exonuclease. Here, we show that the rad50, mre11 and nurA genes from the hyperthermophilic archaeon Sulfolobus acidocaldarius are co-transcribed with a fourth gene encoding a DNA helicase. This enzyme (HerA) is the prototype of a new class of DNA helicases able to utilize either 3' or 5' single-stranded DNA extensions for loading and subsequent DNA duplex unwinding. To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously. Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases. HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together. These results suggest that the archaeal Rad50-Mre11 complex might act in association with a 5' to 3' exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous recombination.     

The Sulfolobus solfataricus RecQ-like DNA helicase Hel112 inhibits the NurA/HerA complex exonuclease activity

ATPase/Helicases and nucleases play important roles in DNA end-resection, a critical step during homologous recombination repair in all organisms. In hyperthermophilic archaea the exo-endonuclease NurA and the ATPase HerA cooperate with the highly conserved Mre11-Rad50 complex in 3′ single-stranded DNA (ssDNA) end processing to coordinate repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA complex. In this study we demonstrate that the NurA exonuclease activity is inhibited by the Sulfolobus solfataricus RecQ-like Hel112 helicase. Inhibition occurs both in the presence and in the absence of HerA, but is much stronger when NurA is in complex with HerA. In contrast, the endonuclease activity of NurA is not affected by the presence of Hel112. Taken together these results suggest that the functional interaction between NurA/HerA and Hel112 is important for DNA end-resection in archaeal homologous recombination.

     

DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex.

Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA double-strand breaks. We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity. Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex. In the Rad50.Mre11 complex, the protein components exist in equimolar amounts. Mre11 has a 3' to 5' exonuclease activity that results in the release of mononucleotides. The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11. Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50.Mre11 is enhanced for substrates with duplex DNA ends. We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3' and 5' single-stranded DNA overhangs. Mre11 is capable of cleaving hairpins and the 3' single-stranded DNA tail. These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP. Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5' strand at secondary structures formed upon DNA strand separation.     

NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing

The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn²⁺), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing.     

Physical and functional interaction between archaeal single-stranded DNA-binding protein and the 5'-3' nuclease NurA

NurA is a novel 5′-3′ exonuclease that is closely linked to Mre11 and Rad50 homologues in most thermophilic archaea. We report a physical and functional interaction between NurA (StoNurA) and single-stranded DNA-binding protein (StoSSB) from the hyperthermophilic archaeon Sulfolobus tokodaii. StoSSB was identified as a novel StoNurA-interacting protein by pull-down assay using Ni-NTA agarose beads and MALDI-TOF mass spectrometry. The direct interaction between StoNurA and StoSSB was further confirmed by yeast two-hybrid and co-immunoprecipitation analysis. The interaction was supposed to have functional significance because it was found that StoSSB inhibited the 5′-3′ ssDNA and dsDNA exonuclease and ssDNA endonuclease activities of StoNurA. Our results suggest that NurA may function closely together with SSB in DNA transactions in archaea.     

NurA,a novel 5'-3' nuclease gene linked to rad50 and mre11 homologs of thermophilic Archaea

We isolated and characterized a new nuclease (NurA) exhibiting both single-stranded endonuclease activity and 5′–3′ exonuclease activity on single-stranded and double-stranded DNA from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Nuclease homologs are detected in all thermophilic archaea and, in most species, the nurA gene is organized in an operon-like structure with rad50 and mre11 archaeal homologs. This nuclease might thus act in concert with Rad50 and Mre11 proteins in archaeal recombination/repair. To our knowledge, this is the first report of a 5′–3′ nuclease potentially associated with Rad50 and Mre11-like proteins that may lead to the processing of double-stranded breaks in 3′ single-stranded tails.     

DNA end resection--unraveling the tail

Homology-dependent repair of DNA double-strand breaks (DSBs) initiates by the 5'-3' resection of the DNA ends to create single-stranded DNA (ssDNA), the substrate for Rad51/RecA binding. Long tracts of ssDNA are also required for activation of the ATR-mediated checkpoint response. Thus, identifying the proteins required and the underlying mechanism for DNA end resection has been an intense area of investigation. Genetic studies in Saccharomyces cerevisiae show that end resection takes place in two steps. Initially, a short oligonucleotide tract is removed from the 5' strand to create an early intermediate with a short 3' overhang. Then in a second step the early intermediate is rapidly processed generating an extensive tract of ssDNA. The first step is dependent on the highly conserved Mre11-Rad50-Xrs2 complex and Sae2, while the second step employs the exonuclease Exo1 and/or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 with the endonuclease Dna2. Here we review recent in vitro and in vivo findings that shed more light into the mechanisms of DSB processing in mitotic and meiotic DSB repair as well as in telomere metabolism.     

Crystal structure of the NurA-dAMP-Mn2+ complex

Generation of the 3′ overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5′–3′ nuclease, NurA, plays an important role in generating 3′ single-stranded DNA during archaeal HR, together with Mre11–Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn²⁺-bound NurA from Pyrococcus furiousus (Pf NurA) to provide the basis for its cleavage mechanism. Pf NurA forms a pyramid-shaped dimer containing a large central channel on one side, which becomes narrower towards the peak of the pyramid. The structure contains a PIWI domain with high similarity to argonaute, endoV nuclease and RNase H. The two active sites, each of which contains Mn²⁺ ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer. The 3′ OH group of the ribose ring is directed toward the channel entrance, explaining the 5′–3′ nuclease activity of Pf NurA. We provide a DNA binding and cleavage model for Pf NurA.     

The characterization of Saccharomyces cerevisiae Mre11/Rad50/Xrs2 complex reveals that Rad50 negatively regulates Mre11 endonucleolytic but not the exonucleolytic activity

The evolutionarily conserved heterotrimeric Mre11/Rad50/Xrs2 (Nbs1) (MRX/N) complex plays a central role in an array of cellular responses involving DNA damage, telomere length homeostasis, cell-cycle checkpoint control and meiotic recombination. The underlying biochemical functions of MRX/N complex, or each of its individual subunits, at telomeres and the importance of complex formation are poorly understood. Here, we show that the Saccharomyces cerevisiae MRX complex, or its subunits, display an overwhelming preference for G-quadruplex DNA than for telomeric single-stranded or double-stranded DNA implicating the possible existence of this DNA structure in vivo. Although these alternative DNA substrates failed to affect Rad50 ATPase activity, kinetic analyses revealed that interaction of Rad50 with Xrs2 and/or Mre11 led to a twofold increase in the rates of ATP hydrolysis. Significantly, we show that Mre11 displays sequence-specific double-stranded DNA endonuclease activity, and Rad50, but not Xrs2, abrogated endonucleolytic but not the exonucleolytic activity. This repression was alleviated upon ATP hydrolysis by Rad50, suggesting that complex formation between Rad50 and Mre11 might be important for blocking the inappropriate cleavage of genomic DNA. Mre11 alone, or in the presence of ATP, MRX, MR or MX sub-complexes cleaved at the 5' end of an array of G residues in single-stranded DNA, at G quartets in G4 DNA, and at the center of TGTG repeats in duplex DNA. We propose that negative regulation of Mre11 endonuclease activity by Rad50 might be important for native as well as de novo telomere length homeostasis.     

The MRX complex regulates Exo1 resection activity by altering DNA end structure

Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double‐strand breaks (DSBs). DSB resection requires the Mre11‐Rad50‐Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11‐R10T mutant variant that accelerates DSB resection compared to wild‐type Mre11 by potentiating Exo1‐mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double‐strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.     

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.     

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.     

Archaeal DNA helicase HerA interacts with Mre11 homologue and unwinds blunt-ended double-stranded DNA and recombination intermediates

HerA is a novel family DNA helicases that exist ubiquitously in thermophilic archaea. The genes are linked to homologues of eukaryotic recombination and repair proteins Mre11 and Rad50 in some of the genomes. However, the relationship between HerA and the related proteins is unclear. In this study, a homologue from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHerA) was characterized and physical and functional interactions between StoHerA and StoMre11 (Mre11 from S. tokodaii) were studied. It was found that StoHerA was able to unwind blunt-ended double-stranded DNA (dsDNA), although with lower efficiency. StoHerA was also able to unwind Holliday junction, splayed-arm DNA, as well as 5'- or 3'-overhang with high efficiency. Pull-down and yeast two-hybrid analyses revealed that StoHerA interacted with StoMre11 physically. The helicase activity of StoHerA was stimulated by StoMre11, indicating a functional role of this interaction. In addition, site-directed mutagenesis of StoHerA was performed to analyze functions of conserved residues of StoHerA. Interestingly, mutation of E355 to alanine in Walker B resulted in not only loss of ATPase and DNA helicase activities, but also dsDNA-binding ability, indicating that this residue is involved in the coupling of ATP hydrolysis, dsDNA-binding, and helicase activities.     

Association of Mre11p with double-strand break sites during yeast meiosis

The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.     

Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).

Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways.     

Structure of the Rad50 DNA double‐strand break repair protein in complex with DNA

The Mre11–Rad50 nuclease–ATPase is an evolutionarily conserved multifunctional DNA double‐strand break (DSB) repair factor. Mre11–Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50^NBD (nucleotide‐binding domain) in complex with Mre11^HLH (helix‐loop‐helix domain), AMPPNP, and double‐stranded DNA. DNA binds between both coiled‐coil domains of the Rad50 dimer with main interactions to a strand‐loop‐helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double‐strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP‐bound state.     

Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection,End-Tethering and Repair of a Double-Strand Break

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.     

Making the best of the loose ends: Mre11/Rad50 complexes and Sae2 promote DNA double-strand break resection

Double-strand breaks in chromosomal DNA are repaired efficiently in eukaryotic cells through pathways that involve direct religation of broken ends, or through pathways that utilize an unbroken, homologous DNA molecule as a template for replication. Pathways of repair that require homology initiate with the resection of the 5' strand at the break site, to uncover the 3' single-stranded DNA that becomes a critical intermediate in single-strand annealing and in homologous strand exchange. Resection of the 5' strand is regulated to occur most efficiently in S and G(2) phases of the cell cycle when sister chromatids are present as recombination templates. The mechanisms governing resection in eukaryotes have been elusive for many years, but recent work has identified the major players in short-range processing of DNA ends as well as the extensive resection of breaks that has been observed in vivo. This review focuses on the Mre11/Rad50/Xrs2(Nbs1) complex and the Sae2(CtIP) protein and their roles in initiating both short-range and long-range resection, the effects of topoisomerase-DNA conjugates on resection in vivo, and the relationship between these factors and NHEJ proteins in regulating 5' strand resection in eukaryotic cells.