The role of Holliday junction resolvases in the repair of spontaneous and induced DNA damage

DNA double-strand breaks (DSBs) and other lesions occur frequently during cell growth and in meiosis. These are often repaired by homologous recombination (HR). HR may result in the formation of DNA structures called Holliday junctions (HJs), which need to be resolved to allow chromosome segregation. Whereas HJs are present in most HR events in meiosis, it has been proposed that in vegetative cells most HR events occur through intermediates lacking HJs. A recent screen in yeast has shown HJ resolution activity for a protein called Yen1, in addition to the previously known Mus81/Mms4 complex. Yeast strains deleted for both YEN1 and MMS4 show a reduction in growth rate, and are very sensitive to DNA-damaging agents. In addition, we investigate the genetic interaction of yen1 and mms4 with mutants defective in different repair pathways. We find that in the absence of Yen1 and Mms4 deletion of RAD1 or RAD52 have no further effect, whereas additional sensitivity is seen if RAD51 is deleted. Finally, we show that yeast cells are unable to carry out meiosis in the absence of both resolvases. Our results show that both Yen1 and Mms4/Mus81 play important (although not identical) roles during vegetative growth and in meiosis.     

Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination.

We have determined the X-ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre recombinase. The HJ is a four-way branched structure that occurs as an intermediate in genetic recombination pathways, including site-specific recombination by the lambda-integrase family. Cre recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre recombinase bound to an immobile HJ and the 2.5 A structure of Cre recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold-symmetric DNA intermediate that shares features with both the stacked-X and the square conformations of the HJ that exist in the unbound state. The structures support a protein-mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of recombinase active sites. In this model, a subtle isomerization of the Cre recombinase-HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.     

Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.     

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.     

Evidence that phenylalanine 69 in Escherichia coli RuvC resolvase forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate

Escherichia coli RuvC resolvase is a specific endonuclease that recognizes and cleaves Holliday junctions formed during homologous recombination and recombinational repair. This study examines the phenotype of RuvC mutants with amino acid substitutions at phenylalanine 69 (F69L, F69Y, F69W, and F69A), a catalytically important residue that faces the catalytic center of the enzyme. F69Y, but not the other three mutants, almost fully complements the UV sensitivity of a DeltaruvC strain and substantially resolves synthetic Holliday junctions in vitro. In the presence of 100 mm NaCl, RuvC F69A and F69L are defective in junction binding, but F69Y and F69W retain near wild-type binding activity during a gel shift binding assay. KMnO(4) was used to probe synthetic Holliday junction DNA in a complex with wild-type and mutant RuvC; F69A and F69L did not induce disruption of base pairing at the crossover to the same extent as wild-type RuvC. Thus, the aromatic ring of Phe-69 is involved in DNA binding, probably via a stacking interaction with a nucleotide base, and this interaction may induce a structural change in junction DNA that is required to form a catalytically competent complex.     

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.     

Saccharomyces cerevisiae Hop1 protein zinc finger motif binds to the Holliday junction and distorts the DNA structure: implications for holliday junction migration

Saccharomyces cerevisiae HOP1, which encodes a component of synaptonemal complex, plays an important role in crossing over between homologues. Hop1p contains a zinc finger motif, and substitution of a conserved Cys371 by Ser rendered the hop1 mutant allele defective in sporulation and meiosis. However, the molecular mechanism underlying the function of Hop1 zinc finger motif (ZnF) remains obscure. Here we show that wild-type Hop1 ZnF binds significantly better to the Holliday junction compared with other recombination intermediates. Consequently, the salt titration midpoint for dissociation of the Holliday junction-ZnF complex was higher than the complexes containing flush-ended linear or tailed duplex DNA. Although DNase I footprinting showed that Hop1 ZnF binds to each of the four arms of the junction, KMnO4 probing and 2-aminopurine fluorescence emission data disclosed that it distorts the DNA structure along a pair of symmetrical arms. Molecular modeling studies show that Hop1 ZnF forms a unique zinc-binding fold, reminiscent of the basic helix-loop-helix motif. In the presence of Zn2+, docking studies show that alpha helix 1, which is replete with basic amino acid residues, makes stabilizing contacts with the sugar-phosphate backbone. Structural comparison revealed a striking similarity between RecG wedge domain and Hop1 ZnF motif. We propose that Hop1 ZnF motif plays a key role in the physical monitoring of recombination intermediates and branch migration of the Holliday junction.     

Resolution of a Holliday junction by vaccinia topoisomerase requires a spacer DNA segment 3' of the CCCTT/ cleavage sites

Vaccinia virus DNA topoisomerase catalyzes reso­lution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5′-CCCTT↓, that are opposed within a partially mobile four-way junction. Efficient resolution occurs on a junction with a 10 bp segment of branch mobility (5′-GCCCTTATCG) that extends 4 bp 3′ of the scissile phosphate. Here we report that resolution is decreased when branch mobility is limited to an 8 bp segment extending 2 bp 3′ of the cleavage site and then eliminated when branch mobility is confined to the 6 bp GCCCTT sequence 5′ of the scissile phosphate. We surmise that a spacer region 3′ of CCCTT is needed for simultaneous cleavage at two opposing sites at the junction. Branch mobility is not required for reaction chem­i­stry at a junction, because topoisomerase cleaves a single CCCTT site in a non-mobile four-way junction where the scissile phosphate is at the crossover point. The junction resolvase activity of topo­isomerase may be involved in forming the hairpin telomeres of the vaccinia genome.     

Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1

Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange. In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques. Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium. The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold. This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments [Overmars and Altona (1997) J. Mol. Biol. 273, 519-524; Panyutin et al. (1995) EMBO J. 14, 1819-1826]. The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.     

Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors

Phospholipase D (PLD) and heterotrimeric G-protein both play important, diverse roles in cellular regulation and signal transduction. Here we have determined the physical interaction between plant PLD and the only canonical alpha-subunit (Galpha) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction. PLDalpha1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with Galpha. PLDalpha1 contains a sequence motif analogous to the G alpha-interacting DRY motif normally conserved in G-protein-coupled receptors. Mutation of the central Lys residue PLD(K564A) of this motif abolished the PLDalpha1-Galpha binding, whereas mutation of the two flanking residues PLD(E563A) and PLD(F565A) decreased the binding. Addition of Galpha to PLDalpha1 inhibited PLDalpha1 activity, whereas the PLD(K564A) mutation that disrupted the Galpha-PLDalpha1 binding abolished the inhibition. GTP relieved the Galpha inhibition of PLDalpha1 activity and also inhibited the binding between PLDalpha1 and Galpha. Meanwhile, the PLDalpha1-Galpha interaction stimulated the intrinsic GTPase activity of Galpha. Therefore, these results have demonstrated the direct binding between Galpha and PLDalpha1, identified the DRY motif on PLDalpha1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLDalpha1 and Galpha.     

Interaction of inhibitor-2 with the catalytic subunit of type 1 protein phosphatase. Identification of a sequence analogous to the consensus type 1 protein phosphatase-binding motif

Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919-28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH(2)-terminal I-2 regions containing residues 1-35 and 1-64 have no inhibitory activity on their own, they increase the IC(50) for I-2 by approximately 30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys(144)-Leu-His-Tyr(147) as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135-151) significantly increases the IC(50) for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135-151) peptide, but not I-2(1-35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, R(GL)/G(M)(61-80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH(2)-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple "anchoring interactions" serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself.     

A DNA invertase from Staphylococcus aureus is a member of the Hin family of site-specific recombinases

Abstract The nucleotide sequence of a staphylococcal plasmid gene has been found to encode a protein highly homologous to the Hin family of conservative site-specific recombination proteins.     

The targeting of cystinosin to the lysosomal membrane requires a tyrosine-based signal and a novel sorting motif

Cystinosis is a lysosomal transport disorder characterized by an accumulation of intra-lysosomal cystine. Biochemical studies showed that the lysosomal cystine transporter was distinct from the plasma membrane cystine transporters and that it exclusively transported cystine. The gene underlying cystinosis, CTNS, encodes a predicted seven-transmembrane domain protein called cystinosin, which is highly glycosylated at the N-terminal end and carries a GY-XX-Phi (where Phi is a hydrophobic residue) lysosomal-targeting motif in its carboxyl tail. We constructed cystinosin-green fluorescent protein fusion proteins to determine the subcellular localization of cystinosin in transfected cell lines and showed that cystinosin-green fluorescent protein colocalizes with lysosomal-associated membrane protein 2 (LAMP-2) to lysosomes. Deletion of the GY-XX-Phi motif resulted in a partial redirection to the plasma membrane as well as sorting to lysosomes, demonstrating that this motif is only partially responsible for the lysosomal targeting of cystinosin and suggesting the existence of a second sorting signal. A complete relocalization of cystinosin to the plasma membrane was obtained after deletion of half of the third cytoplasmic loop (amino acids 280-288) coupled with the deletion of the GY-DQ-L motif, demonstrating the presence of the second signal within this loop. Using site-directed mutagenesis studies we identified a novel conformational lysosomal-sorting motif, the core of which was delineated to YFPQA (amino acids 281-285).     

Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.     

Receipt of the C-terminal tail from a neighboring lambda Int protomer allosterically stimulates Holliday junction resolution

Bacteriophage lambda integrase (Int) catalyzes the integration and excision of the phage lambda chromosome into and out of the Esherichia coli host chromosome. The seven carboxy-terminal residues (C-terminal tail) of Int comprise a context-sensitive regulatory element that links catalytic function with protein multimerization and also coordinates Int functions within the multimeric recombinogenic complex. The experiments reported here show that the beta5-strand of Int is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail. Using a mutant integrase in which the C-terminal tail has been deleted (W350ter), we demonstrate that the C-terminal tail is required for efficient and accurate resolution of Holliday junctions by tetrameric Int. Addition of a free heptameric peptide of the same sequence as the C-terminal tail partially reverses the W350ter defects by stimulating Holliday junction resolution. The peptide also stimulates the topoisomerase function of monomeric W350ter. Single residue alterations in the peptide sequence and a mutant of the beta5 strand indicate that the observed stimulation arises from specific contacts with the beta5 strand (residues 239-243). The peptide does not stimulate binding of W350ter to its cognate DNA sites and therefore appears to recapitulate the effects of the normal C-terminal tail intermolecular contacts in wild-type Int. Models for the allosteric stimulation of Int activity by beta5 strand contacts are discussed.     

Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution

The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration

Toxigenic conversion of Vibrio cholerae bacteria results from the integration of a filamentous phage, CTXϕ. Integration is driven by the bacterial Xer recombinases, which catalyse the exchange of a single pair of strands between the phage single-stranded DNA and the host double-stranded DNA genomes; replication is thought to convert the resulting pseudo-Holliday junction (HJ) intermediate into the final recombination product. The natural tendency of the Xer recombinases to recycle HJ intermediates back into substrate should thwart this integration strategy, which prompted a search for additional co-factors aiding directionality of the process. Here, we show that Endo III, a ubiquitous base excision repair enzyme, facilitates CTXϕ-integration in vivo. In vitro, we show that it prevents futile Xer recombination cycles by impeding new rounds of strand exchanges once the pseudo-HJ is formed. We further demonstrate that this activity relies on the unexpected ability of Endo III to bind to HJs even in the absence of the recombinases. These results explain how tandem copies of the phage genome can be created, which is crucial for subsequent virion production.