Effect of sequence on the conformation of DNA holliday junctions

Structures of the DNA sequences d(CCGGCGCCGG) and d(CCAGTACbr(5)UGG) are presented here as four-way Holliday junctions in their compact stacked-X forms, with antiparallel alignment of the DNA strands. Thus, the ACC-trinucleotide motif, previously identified as important for stabilizing the junction, is now extended to PuCPy, where Pu is either an adenine or guanine, and Py is either a cytosine, 5-methylcytosine, or 5-bromouracil but not thymine nucleotide. We see that both sequence and base substituents affect the geometry of the junction in terms of the interduplex angle as well as the previously defined conformational variables, J(roll) (the rotation of the stacked duplexes about their respective helical axis) and J(slide) (the translational displacement of the stacked duplexes along their respective helical axis). The structures of the GCC and parent ACC containing junctions fall into a distinct conformational class that is relatively undistorted in terms of J(slide) and J(roll), with interduplex angles of 40-43 degrees. The substituted ACbr(5)U structure, however, is more akin to that of the distorted methylated ACm(5)C containing junction, with J(slide) (>or=2.3 A) and a similar J(roll) (164 degrees) opening the major groove-side of the junction, but shows a reduced interduplex angle. In contrast, the analogous d(CCAGTACTGG) sequence has to date been crystallized only as resolved B-DNA duplexes. This suggests that there is an electronic effect of substituents at the pyrimidine Py position on the stability of four-stranded junctions. The single-crystal structures presented here, therefore, show how sequence affects the detailed geometry, and subsequently, the associated stability and conformational dynamics of the Holliday junction.     

Mechanism of spontaneous DNA-DNA interaction of homologous linear duplexes

Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.     

Specificity of binding to four-way junctions in DNA by bacteriophage T7 endonuclease I.

T7 endonuclease I binds specifically to four-way junctions in duplex DNA and promotes their resolution into linear duplexes. Under conditions in which the nuclease activity is blocked by the absence of divalent cations, the enzyme forms a distinct protein-DNA complex with the junction, as detected by gel retardation and filter binding assays. The formation of this complex is structure-specific and contrasts with the short-lived binding complexes formed on linear duplex DNA. The binding complex between T7 endonuclease I and a synthetic Holliday junction analog has been probed with hydroxyl radicals. The results indicate that the nuclease binds all four strands about the junction point.     

The X philes: structure-specific endonucleases that resolve Holliday junctions

Genetic recombination is a critical cellular process that promotes evolutionary diversity, facilitates DNA repair and underpins genome duplication. It entails the reciprocal exchange of single strands between homologous DNA duplexes to form a four-way branched intermediate commonly referred to as the Holliday junction. DNA molecules interlinked in this way have to be separated in order to allow normal chromosome transmission at cell division. This resolution reaction is mediated by structure-specific endonucleases that catalyse dual-strand incision across the point of strand cross-over. Holliday junctions can also arise at stalled replication forks by reversing the direction of fork progression and annealing of nascent strands. Resolution of junctions in this instance generates a DNA break and thus serves to initiate rather than terminate recombination. Junction resolvases are generally small, homodimeric endonucleases with a high specificity for branched DNA. They use a metal-binding pocket to co-ordinate an activated water molecule for phosphodiester bond hydrolysis. In addition, most junction endonucleases modulate the structure of the junction upon binding, and some display a preference for cleavage at specific nucleotide target sequences. Holliday junction resolvases with distinct properties have been characterized from bacteriophages (T4 endo VII, T7 endo I, RusA and Rap), Bacteria (RuvC), Archaea (Hjc and Hje), yeast (CCE1) and poxviruses (A22R). Recent studies have brought about a reappraisal of the origins of junction-specific endonucleases with the discovery that RuvC, CCE1 and A22R share a common catalytic core.     

A method for preparing genomic DNA that restrains branch migration of Holliday junctions

The Holliday junction is a central intermediate in genetic recombination. This four-stranded DNA structure is capable of spontaneous branch migration, and is lost during standard DNA extraction protocols. In order to isolate and characterize recombination intermediates that contain Holliday junctions, we have developed a rapid protocol that restrains branch migration of four-way DNA junctions. The cationic detergent hex-adecyltrimethylammonium bromide is used to lyse cells and precipitate DNA. Manipulations are performed in the presence of the cations hexamine cobalt(III) or magnesium, which stabilize Holliday junctions in a stacked-X configuration that branch migrates very slowly. This protocol was evaluated using a sensitive assay for spontaneous branch migration, and was shown to preserve both artificial Holliday junctions and meiotic recombination intermediates containing four-way junctions.     

Analysis of branched nucleic acid structure using comparative gel electrophoresis

Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.     

Gel electrophoretic analysis of the geometry of a DNA four-way junction

Branched DNA molecules (Holliday structures) are believed to be key intermediates in the process of homologous genetic recombination. However, despite the importance of such structures, their transient nature makes it difficult to analyze their physical properties. In an effort to evaluate several models for the geometry of such branched molecules, a stable, synthetic DNA four-way junction has been constructed. The geometry of the synthetic junction has been probed by gel electrophoresis, utilizing the fact that bent DNA molecules demonstrate reduced mobilities on polyacrylamide gels to an extent that varies with the degree of the bend angle. From the synthetic four-way junction, we have produced a set of molecules in which all combinations of two junction arms have been extended by 105 base-pairs. The electrophoretic mobilities of the extended junctions differ in a manner which indicates that the junction is not a completely flexible structure; nor is it tetrahedral or planar-tetragonal. Instead, the four strands that comprise the DNA four-way junction are structurally non-equivalent. The significance of these observations with regard to previous models for four-way junction geometry is discussed.     

Solution formation of Holliday junctions in inverted-repeat DNA sequences

The structure of Holliday junctions has now been well characterized at the atomic level through single-crystal X-ray diffraction in symmetric (inverted-repeat) DNA sequences. At issue, however, is whether the formation of these four-stranded complexes in solution is truly sequence dependent in the manner proposed or is an artifact of the crystallization process and, therefore, has no relevance to the behavior of this central intermediate in homologous recombination and recombination-dependent cellular processes. Here, we apply analytical ultracentrifugation to demonstrate that the sequence d(CCGGTACCGG), which crystallizes in the stacked-X form of the junction, assembles into four-stranded junctions in solution in a manner that is dependent on the DNA and cation concentrations, with an equilibrium established between the junction and duplex forms at 100-200 microM DNA duplex. In contrast, the sequence d(CCGCTAGCGG), which has been crystallized as B-DNA, is seen to adopt only the double-helical form at all DNA and salt concentrations that were tested. Thus, the ACC trinucleotide core is now shown to be important for the formation of Holliday junctions in both crystals and in solution and can be estimated to contribute approximately -4 kcal/mol to stabilizing this recombination intermediate in inverted-repeat sequences.     

Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1

In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg²⁺ ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg²⁺ ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3′-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.     

Crystal structure of a DNA Holliday junction.

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 A resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.     

Crystal structure of the B-DNA hexamer d(CTCGAG): model for an A-to-B transition.

The crystal structure of the B-DNA hexamer d(CTCGAG) has been solved at 1.9 A resolution by iterative single isomorphous replacement, using the brominated derivative d(CG5BrCGAG), and refined to an R-factor of 18.6% for 120 nonhydrogen nucleic acid atoms and 32 water molecules. Although the central four base pairs form a typical B-form helix, several parameters suggest a transition to an A-like conformation at the termini. Based on this observation, a B-to-A transition was modeled, maintaining efficient base stacking across the junction. The wide minor groove (approximately 6.9 A) is reminiscent of that in the side-by-side double drug-DNA complexes and hosts a double spine of hydration. The global helix axes of the pseudo-continuous helices are at an acute angle of 60 degrees. The pseudocontinuous stacking is reinforced by the minor groove water structure extending between the two duplexes. The crossover point of two pairs of stacked duplexes is at the stacking junction, unlike that observed in the B-DNA decamers and dodecamers. This arrangement may have implications for the structure of a four-way DNA junction. The duplexes are arranged around a large (approximately 20 A diameter) channel centered on a 6(2) screw axis.     

The role of the SAP motif in promoting Holliday junction binding and resolution by SpCCE1

Holliday junctions are four-way branched DNA structures that are formed during recombination and by replication fork regression. Their processing depends on helicases that catalyze junction branch migration, and endonucleases that resolve the junction into nicked linear DNAs. Here we have investigated the role of a DNA binding motif called SAP in binding and resolving Holliday junctions by the fission yeast mitochondrial resolvase SpCCE1. Mutation or partial/complete deletion of the SAP motif dramatically impairs the ability of SpCCE1 to resolve Holliday junctions in a heterologous in vivo system. These mutant proteins retain the ability to recognize the junction structure and to distort it upon binding. However, once formed the mutant protein-junction complexes are relatively unstable and dissociate much faster than wild-type complexes. We show that binding stability is necessary for efficient junction resolution, and that this may be due in part to a requirement for maintaining the junction in an open conformation so that it can branch migrate to cleavable sites.     

Resolving Holliday junctions with Escherichia coli UvrD helicase

The Escherichia coli UvrD helicase is known to function in the mismatch repair and nucleotide excision repair pathways and has also been suggested to have roles in recombination and replication restart. The primary intermediate DNA structure in these two processes is the Holliday junction. UvrD has been shown to unwind a variety of substrates including partial duplex DNA, nicked DNA, forked DNA structures, blunt duplex DNA and RNA-DNA hybrids. Here, we demonstrate that UvrD also catalyzes the robust unwinding of Holliday junction substrates. To characterize this unwinding reaction we have employed steady-state helicase assays, pre-steady-state rapid quench helicase assays, DNaseI footprinting, and electron microscopy. We conclude that UvrD binds initially to the junction compared with binding one of the blunt ends of the four-way junction to initiate unwinding and resolves the synthetic substrate into two double-stranded fork structures. We suggest that UvrD, along with its mismatch repair partners, MutS and MutL, may utilize its ability to unwind Holliday junctions directly in the prevention of homeologous recombination. UvrD may also be involved in the resolution of stalled replication forks by unwinding the Holliday junction intermediate to allow bypass of the blockage.     

The endonuclease Hje catalyses rapid, multiple turnover resolution of Holliday junctions

Holliday junction-resolving enzymes are ubiquitous, structure-specific endonucleases that resolve four-way DNA junctions by the introduction of paired nicks in opposing strands, and are required for homologous recombination, double-strand break repair, recombination-dependent restart of stalled or collapsed DNA replication forks, and phage DNA processing. Here, we present the first steady-state kinetic characterisation of a junction-resolving enzyme; the Hje endonuclease from Sulfolobus solfataricus. We demonstrate that substrate turnover by Hje is sequence-independent and limited largely by the rate of cleavage of the phosphodiester bonds of the bound Holliday junction substrate, rather than substrate association or product dissociation. Reaction rates under multiple turnover conditions compare favourably with type II restriction enzymes. These properties, coupled with a high level of specificity for four-way junctions over all other DNA substrates, make Hje a suitable enzyme for applications requiring the detection and cleavage of Holliday junctions in vitro.     

DNA recombination: holliday junctions dynamics and branch migration

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.     

Structures of helical junctions in nucleic acids

Our knowledge of the architectural principles of nucleic acid junctions has seen significant recent advances. The conformation of DNA junctions is now well understood, and this provides a new basis for the analysis of important structural elements in RNA. The most significant new data have come from X-ray crystallography of four-way DNA junctions; incidentally showing the great importance of serendipity in science, since none of the three groups had deliberately set out to crystallise a junction. Fortunately the results confirm, and of course extend, the earlier conformational studies of DNA junctions in almost every detail. This is important, because it means that these methods can be applied with greater confidence to new systems, especially in RNA. Methods like FRET, chemical probing and even the humble polyacrylamide gel can be rapid and very powerful, allowing the examination of a large number of sequence variants relatively quickly. Molecular modelling in conjunction with experiments is also a very important component of the general approach. Ultimately crystallography provides the gold standard for structural analysis, but the other, simple approaches have considerable value along the way. At the beginning of this review I suggested two simple folding principles for branched nucleic acids, and it is instructive to review these in the light of recent data. In brief, these were the tendency for pairwise coaxial stacking of helical arms, and the importance of metal ion interactions in the induction of folding. We see that both are important in a wide range of systems, both in DNA and RNA. The premier example is the four-way DNA junction, which undergoes metal ion-induced folding into the stacked X-structure that is based on coaxial stacking of arms. As in many systems, there are two alternative ways to achieve this depending on the choice of stacking partners. Recent data reveal that both forms often exist in a dynamic equilibrium, and that the relative stability of the two conformers depends upon base sequence extending a significant distance from the junction. The three-way junction has provided a good test of the folding principles. Perfect three-way (3H) DNA junctions seem to defy these principles in that they appear reluctant to undergo coaxial stacking of arms, and exhibit little change in conformation with addition of metal ions. Modelling suggests that such a junction is stereochemically constrained in an extended conformation. However, upon inclusion of a few additional base pairs at the centre (to create a 3HS2 junction for example) the additional stereochemical flexibility allows two arms to undergo coaxial stacking. Such a junction exhibits all the properties consistent with the general folding principles, with ion-induced folding into a form based on pairwise coaxial stacking of arms in one of two different conformers. The three-way junction is therefore very much the exception that proves the rule. It is instructive to compare the folding of corresponding species in DNA and RNA, where we find both similarities and differences. The RNA four-way junction can adopt a structure that is globally similar to the stacked X-structure (Duckett et al. 1995a), and the crystal structure of the DNAzyme shows that the stacked X-conformation can include one helical pair in the A-conformation (Nowakowski et al. 1999). However, modelling suggests that the juxtaposition of strands and grooves will be less satisfactory in RNA, and the higher magnesium ion concentration required to fold the RNA junction indicates a lower stability of the antiparallel form. Perhaps the biggest difference between the properties of the DNA and RNA four-way junctions is the lack of an unstacked structure at low salt concentrations for the RNA species. This clearly reflects a major difference in the electrostatic interactions in the RNA junction. In general the folding of branched DNA provides some good indications on the likely folding of the corresponding RNA species, but caution is required in making the extrapolation because the two polymers are significantly different. A number of studies point to the flexibility and malleability of branched nucleic acids, and this turns out to have particular significance in their interactions with proteins. Proteins such as the DNA junction-resolving enzymes exhibit considerable selectivity for the structure of their substrates, which is still not understood at a molecular level. Despite this, it appears to be universally true that these proteins distort the global, and in some cases at least the local, structure of the junctions. The somewhat perplexing result is that the proteins appear to distort the very property that they recognise. In general it seems that four-way DNA junctions are opened to one extent or another by interaction with proteins. (ABSTRACT TRUNCATED)     

Holliday junction resolution is modulated by archaeal chromatin components in vitro.

The Holliday junction-resolving enzyme Hjc is conserved in the archaea and probably plays a role analogous to that of Escherichia coli RuvC in the pathway of homologous recombination. Hjc specifically recognizes four-way DNA junctions, cleaving them without sequence preference to generate recombinant DNA duplex products. Hjc imposes an X-shaped global conformation on the bound DNA junction and distorts base stacking around the point of cleavage, three nucleotides 3' of the junction center. We show that Hjc is autoinhibitory under single turnover assay conditions and that this can be relieved by the addition of either competitor duplex DNA or the architectural double-stranded DNA-binding protein Sso7d (i.e. by approximating in vivo conditions more closely). Using a combination of isothermal titration calorimetry and fluorescent resonance energy transfer, we demonstrate that multiple Hjc dimers can bind to each synthetic four-way junction and provide evidence for significant distortion of the junction structure at high protein:DNA ratios. Analysis of crystal packing interactions in the crystal structure of Hjc suggests a molecular basis for this autoinhibition. The wider implications of these findings for the quantitative study of DNA-protein interactions is discussed.     

Holliday junction binding and resolution by the Rap structure-specific endonuclease of phage lambda

Rap endonuclease targets recombinant joint molecules arising from phage lambda Red-mediated genetic exchange. Previous studies revealed that Rap nicks DNA at the branch point of synthetic Holliday junctions and other DNA structures with a branched component. However, on X junctions incorporating a three base-pair core of homology or with a fixed crossover, Rap failed to make the bilateral strand cleavages characteristic of a Holliday junction resolvase. Here, we demonstrate that Rap can mediate symmetrical resolution of 50 bp and chi Holliday structures containing larger homologous cores. On two different mobile 50 bp junctions Rap displays a weak preference for cleaving the phosphodiester backbone between 5'-GC dinucleotides. The products of resolution on both large and small DNA substrates can be sealed by T4 DNA ligase, confirming the formation of nicked duplexes. Rap protein was also assessed for its capacity to influence the global conformation of junctions in the presence or absence of magnesium ions. Unlike the known Holliday junction binding proteins, Rap does not affect the angle of duplex arms, implying an unorthodox mode of junction binding. The results demonstrate that Rap can function as a Holliday junction resolvase in addition to eliminating other branched structures that may arise during phage recombination.     

Assembly and characterization of five-arm and six-arm DNA branched junctions.

DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.     

Human Mus81-associated endonuclease cleaves Holliday junctions in vitro.

Mus81, a protein with homology to the XPF subunit of the ERCC1-XPF endonuclease, is important for replicational stress tolerance in both budding and fission yeast. Human Mus81 has associated endonuclease activity against structure-specific oligonucleotide substrates, including synthetic Holliday junctions. Mus81-associated endonuclease resolves Holliday junctions into linear duplexes by cutting across the junction exclusively on strands of like polarity. In addition, Mus81 protein abundance increases in cells following exposure to agents that block DNA replication. Taken together, these findings suggest a role for Mus81 in resolving Holliday junctions that arise when DNA replication is blocked by damage or by nucleotide depletion. Mus81 is not related by sequence to previously characterized Holliday junction resolving enzymes, and it has distinct enzymatic properties that suggest it uses a novel enzymatic strategy to cleave Holliday junctions.