The RecU Holliday junction resolvase acts at early stages of homologous recombination

Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A and RecUR71A do not exert a negative effect on the RecA dATPase and fail to interact with it. Both activities are important in vivo since RecU mutants impaired only in RecA interaction are as sensitive to DNA damaging agents as a deletion mutant.     

The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec⁺ cells, only the full-length RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible N-terminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB·ATPγS·Mg²⁺ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecA-mediated DNA strand exchange. This study shows that the flexible N-terminus of RecU is essential for protein activity.     

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).     

Interaction of Branch Migration Translocases with the Holliday Junction-resolving Enzyme and Their Implications in Holliday Junction Resolution

Double-strand break repair involves the formation of Holliday junction (HJ) structures that need to be resolved to promote correct replication and chromosomal segregation. The molecular mechanisms of HJ branch migration and/or resolution are poorly characterized in Firmicutes. Genetic evidence suggested that the absence of the RuvAB branch migration translocase and the RecU HJ resolvase is synthetically lethal in Bacillus subtilis, whereas a recU recG mutant was viable. In vitro RecU, which is restricted to bacteria of the Firmicutes phylum, binds HJs with high affinity. In this work we found that RecU does not bind simultaneously with RecG to a HJ. RuvB by interacting with RecU bound to the central region of HJ DNA, loses its nonspecific association with DNA, and re-localizes with RecU to form a ternary complex. RecU cannot stimulate the ATPase or branch migration activity of RuvB. The presence of RuvB·ATPγS greatly stimulates RecU-mediated HJ resolution, but the addition of ATP or RuvA abolishes this stimulatory effect. A RecU·HJ·RuvAB complex might be formed. RecU does not increase the RuvAB activities but slightly inhibits them.     

Mus81 cleavage of Holliday junctions: a failsafe for processing meiotic recombination intermediates?

The Holliday junction (HJ) is a central intermediate of homologous recombination. Its cleavage is critical for the formation of crossover recombinants during meiosis, which in turn helps to establish chiasmata and promote genetic diversity. Enzymes that cleave HJs, called HJ resolvases, have been identified in all domains of life except eukaryotic nuclei. Controversially, the Mus81-Eme1 endonuclease has been proposed to be an example of a eukaryotic nuclear resolvase. However, hitherto little or no HJ cleavage has been detected in recombinant preparations of Mus81-Eme1. Here, we report the purification of active forms of recombinant Schizosaccharomyces pombe Mus81-Eme1 and Saccharomyces cerevisiae Mus81-Mms4, which display robust HJ cleavage in vitro, which, in the case of Mus81-Eme1, is as good as the archetypal HJ resolvase RuvC in single turnover kinetic analysis. We also present genetic evidence that suggests that this activity might be utilised as a back-up to Mus81-Eme1's main activity of cleaving nicked HJs during meiosis in S. pombe.     

Combinatorial Regulation of Meiotic Holliday Junction Resolution in C. elegans by HIM-6 (BLM) Helicase,SLX-4, and the SLX-1, MUS-81 and XPF-1 Nucleases

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of “univalents” linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.     

Identification of amino acid residues critical for catalysis of Holliday junction resolution by Mycoplasma genitalium RecU

The RecU protein from Mycoplasma genitalium, RecU(Mge), is a 19.4-kDa Holliday junction (HJ) resolvase that binds in a nonspecific fashion to HJ substrates and, in the presence of Mn(2+), cleaves these substrates at a specific sequence (5'-G/TC↓C/TTA/GG-3'). To identify amino acid residues that are crucial for HJ binding and/or cleavage, we generated a series of 16 deletion mutants (9 N- and 7 C-terminal deletion mutants) and 31 point mutants of RecU(Mge). The point mutations were introduced at amino acid positions that are highly conserved among bacterial RecU-like sequences. All mutants were purified and tested for the ability to bind to, and cleave, HJ substrates. We found the five N-terminal and three C-terminal amino acid residues of RecU(Mge) to be dispensable for its catalytic activities. Among the 31 point mutants, 7 mutants were found to be inactive in both HJ binding and cleavage. Interestingly, in 12 other mutants, these two activities were uncoupled; while these proteins displayed HJ-binding characteristics similar to those of wild-type RecU(Mge), they were unable to cleave HJ substrates. Thus, 12 amino acid residues were identified (E11, K31, D57, Y58, Y66, D68, E70, K72, T74, K76, Q88, and L92) that may play either a direct or indirect role in the catalysis of HJ resolution.     

Bacillus subtilis RecU Holliday-junction resolvase modulates RecA activities

The Bacillus subtilis RecU protein is able to catalyze in vitro DNA strand annealing and Holliday-junction resolution. The interaction between the RecA and RecU proteins, in the presence or absence of a single-stranded binding (SSB) protein, was studied. Substoichiometric amounts of RecU enhanced RecA loading onto single-stranded DNA (ssDNA) and stimulated RecA-catalyzed D-loop formation. However, RecU inhibited the RecA-mediated three-strand exchange reaction and ssDNA-dependent dATP or rATP hydrolysis. The addition of an SSB protein did not reverse the negative effect exerted by RecU on RecA function. Annealing of circular ssDNA and homologous linear 3′-tailed double-stranded DNA by RecU was not affected by the addition of RecA both in the presence and in the absence of SSB. We propose that RecU modulates RecA activities by promoting RecA-catalyzed strand invasion and inhibiting RecA-mediated branch migration, by preventing RecA filament disassembly, and suggest a potential mechanism for the control of resolvasome assembly.     

Meiotic DNA joint molecule resolution depends on Nse5�CNse6 of the Smc5�CSmc6 holocomplex

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81–Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81–Eme1 is unclear. In cells lacking Nse5–Nse6 of the Smc5–Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5–Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5–Smc6 complex in HJ resolution via Mus81–Eme1.     

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.     

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.     

Holliday junction resolution: Regulation in space and time

Holliday junctions (HJs) can be formed between sister chromatids or homologous chromosomes during the recombinational repair of DNA lesions. A variety of pathways act upon HJs to remove them from DNA, in events that are critical for appropriate chromosome segregation. Despite the identification and characterization of multiple enzymes involved in HJ processing, the cellular mechanisms that regulate and implement pathway usage have only just started to be delineated. A conserved network of core cell-cycle kinases and phosphatases modulate HJ metabolism by exerting spatial and temporal control over the activities of two structure-selective nucleases: yeast Mus81-Mms4 (human MUS81-EME1) and Yen1 (human GEN1). These regulatory cycles operate to establish the sequential activation of HJ processing enzymes, implementing a hierarchy in pathway usage that ensure the elimination of chromosomal interactions which would otherwise interfere with chromosome segregation. Mus81-Mms4/EME1 and Yen1/GEN1 emerge to define a special class of enzymes, evolved to satisfy the cellular need of safeguarding the completion of DNA repair when on the verge of chromosome segregation.     

ruvA Mutants that resolve Holliday junctions but do not reverse replication forks

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination.     

Small molecule functional analogs of peptides that inhibit λ site-specific recombination and bind Holliday junctions

Our lab has isolated hexameric peptides that are structure-selective ligands of Holliday junctions (HJ), central intermediates of several DNA recombination reactions. One of the most potent of these inhibitors, WRWYCR, has shown antibacterial activity in part due to its inhibition of DNA repair proteins. To increase the therapeutic potential of these inhibitors, we searched for small molecule inhibitors with similar activities. We screened 11 small molecule libraries comprising over nine million individual compounds and identified a potent N-methyl aminocyclic thiourea inhibitor that also traps HJs formed during site-specific recombination reactions in vitro. This inhibitor binds specifically to protein-free HJs and can inhibit HJ resolution by RecG helicase, but only showed modest growth inhibition of bacterial with a hyperpermeable outer membrane; nonetheless, this is an important step in developing a functional analog of the peptide inhibitors.     

Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution

During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to selectively stabilize the "activated" phosphate conformation in order to promote cleavage.     

Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae

In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Δ mutants are repair-proficient, yen1Δ mus81Δ double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Δ mus81Δ double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage.     

Evidence for Different Pathways during Horizontal Gene Transfer in Competent Bacillus subtilis Cells

Cytological and genetic evidence suggests that the Bacillus subtilis DNA uptake machinery localizes at a single cell pole and takes up single-stranded (ss) DNA. The integration of homologous donor DNA into the recipient chromosome requires RecA, while plasmid establishment, which is independent of RecA, requires at least RecO and RecU. RecA and RecN colocalize at the polar DNA uptake machinery, from which RecA forms filamentous structures, termed threads, in the presence of chromosomal DNA. We show that the transformation of chromosomal and of plasmid DNA follows distinct pathways. In the absence of DNA, RecU accumulated at a single cell pole in competent cells, dependent on RecA. Upon addition of any kind of DNA, RecA formed highly dynamic thread structures, which rapidly grew and shrank, and RecU dissipated from the pole. RecO visibly accumulated at the cell pole only upon addition of plasmid DNA, and, to a lesser degree, of phage DNA, but not of chromosomal DNA. RecO accumulation was weakly influenced by RecN, but not by RecA. RecO annealed ssDNA complexed with SsbA in vitro, independent of any nucleotide cofactor. The DNA end-joining Ku protein was also found to play a role in viral and plasmid transformation. On the other hand, transfection with SPP1 phage DNA required functions from both chromosomal and plasmid transformation pathways. The findings show that competent bacterial cells possess a dynamic DNA recombination machinery that responds in a differential manner depending if entering DNA shows homology with recipient DNA or has self-annealing potential. Transformation with chromosomal DNA only requires RecA, which forms dynamic filamentous structures that may mediate homology search and DNA strand invasion. Establishment of circular plasmid DNA requires accumulation of RecO at the competence pole, most likely mediating single-strand annealing, and RecU, which possibly down-regulates RecA. Transfection with SPP1 viral DNA follows an intermediate route that contains functions from both chromosomal and plasmid transformation pathways.     

Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution

Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the ‘nick-counter-nick’ mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.     

GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism

Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases.     

The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism

Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.