Holliday Junction Resolvase Research Articles

Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes—T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V—have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products.

Specific Binding of Poly(ADP-ribose) Polymerase-1 to Cruciform Hairpins

Poly(ADP-ribose) polymerase-1 (PARP-1) participates in DNA cleavage and rejoining-dependent reactions, such as DNA replication, recombination and repair. PARP-1 is also important in transcriptional regulation, although the determinants for its binding to undamaged genomic DNA have not been defined. Previously, we have shown by low-resolution mapping that PARP-1 may bind to the cruciform-forming regions of its own promoter. Here, using DNase I and nuclease P(1) footprinting and atomic force microscopy, we show that PARP-1 binds to stem/loop boundaries of cruciform hairpins. Cleavage of the cruciform by the junction resolvase T4 endonuclease VII is independent of PARP-1, which indicates that PARP-1 does not bind to the four-arm junctions of the cruciform. Thus, PARP-1 differs from other cruciform-binding proteins by binding to hairpin tips rather than to junctions. Furthermore, our data indicate that PARP-1 can interact with the gene regulatory sequences by binding to the promoter-localized cruciforms.

Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site

BackgroundPrediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636.ResultsUsing protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family.ConclusionAfter submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.

Evolution of a phage RuvC endonuclease for resolution of both Holliday and branched DNA junctions

Resolution of Holliday junction recombination intermediates in most Gram-negative bacteria is accomplished by the RuvC endonuclease acting in concert with the RuvAB branch migration machinery. Gram-positive species, however, lack RuvC, with the exception of distantly related orthologues from bacteriophages infecting Lactococci and Streptococci. We have purified one of these proteins, 67RuvC, from Lactococcus lactis phage bIL67 and demonstrated that it functions as a Holliday structure resolvase. Differences in the sequence selectivity of resolution between 67RuvC and Escherichia coli RuvC were noted, although both enzymes prefer to cleave 3' of thymidine residues. However, unlike its cellular counterpart, 67RuvC readily binds and cleaves a variety of branched DNA substrates in addition to Holliday junctions. Plasmids expressing 67RuvC induce chromosomal breaks, probably as a consequence of replication fork cleavage, and cannot be recovered from recombination-defective E. coli strains. Despite these deleterious effects, 67RuvC constructs suppress the UV light sensitivity of ruvA, ruvAB and ruvABC mutant strains confirming that the phage protein mediates Holliday junction resolution in vivo. The characterization of 67RuvC offers a unique insight into how a Holliday junction-specific resolvase can evolve into a debranching endonuclease tailored to the requirements of phage recombination.

The DNA repair helicase UvrD is essential for replication fork reversal in replication mutants.

Replication forks arrested by inactivation of the main Escherichia coli DNA polymerase (polymerase III) are reversed by the annealing of newly synthesized leading- and lagging-strand ends. Reversed forks are reset by the action of RecBC on the DNA double-strand end, and in the absence of RecBC chromosomes are linearized by the Holliday junction resolvase RuvABC. We report here that the UvrD helicase is essential for RuvABC-dependent chromosome linearization in E. coli polymerase III mutants, whereas its partners in DNA repair (UvrA/B and MutL/S) are not. We conclude that UvrD participates in replication fork reversal in E. coli.

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast.

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.

Genetic recombination in Bacillus subtilis 168: contribution of Holliday junction processing functions in chromosome segregation.

Bacillus subtilis mutants classified within the epsilon (ruvA, DeltaruvB, DeltarecU, and recD) and eta (DeltarecG) epistatic groups, in an otherwise rec+ background, render cells impaired in chromosomal segregation. A less-pronounced segregation defect in DeltarecA and Deltasms (DeltaradA) cells was observed. The repair deficiency of addAB, DeltarecO, DeltarecR, recH, DeltarecS, and DeltasubA cells did not correlate with a chromosomal segregation defect. The sensitivity of epsilon epistatic group mutants to DNA-damaging agents correlates with ongoing DNA replication at the time of exposure to the agents. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect in ruvA and recD cells and the segregation defect in ruvA and DeltarecG cells. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect of DeltarecU cells but do not suppress the segregation defect in these cells. The DeltarecA mutation suppresses the segregation defect but does not suppress the DNA repair defect in DeltarecU cells. These results result suggest that (i) the RuvAB and RecG branch migrating DNA helicases, the RecU Holliday junction (HJ) resolvase, and RecD bias HJ resolution towards noncrossovers and that (ii) Sms (RadA) and SubA proteins might play a role in the stabilization and or processing of HJ intermediates.

Holliday Junction Binding and Resolution by the Rap Structure-specific Endonuclease of Phage λ

Rap endonuclease targets recombinant joint molecules arising from phage λ 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 χ 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.

Cells defective for replication restart undergo replication fork reversal.

We have studied the fate of blocked replication forks with the use of the Escherichia coli priA mutant, in which spontaneously arrested replication forks persist owing to the lack of the major replication restart pathway. Such blocked forks undergo a specific reaction named replication fork reversal, in which newly synthesized strands anneal to form a DNA double-strand end adjacent to a four-way junction. Indeed, (i) priA recB mutant chromosomes are linearized by a reaction that requires the presence of the Holliday junction resolvase RuvABC, and (ii) RuvABC-dependent linearization is prevented by the presence of RecBC. Replication fork reversal in a priA mutant occurs independently of the recombination proteins RecA and RecR. recBC inactivation does not affect priA mutant viability but prevents priA chronic SOS induction. We propose that, in the absence of PriA, RecBC action at reversed forks does not allow replication restart, which leads to the accumulation of SOS-inducing RecA filaments. Our results suggest that types of replication blockage that cause replication fork reversal occur spontaneously.

RecG helicase promotes DNA double-strand break repair.

Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood. To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA.

Methods for analysis of poxvirus DNA replication.

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polymerase (E9), an essential and stoichiometric component of the processive polymerase (A20), a single-strand DNA-binding protein (I3), a type I topoisomerase (H6), the uracil DNA glycosylase (D4), a nucleic acid-independent nucleoside triphosphatase (D5), a serine/threonine protein kinase (B1), and a Holliday Junction resolvase (A22). All of these factors work in concert to faithfully duplicate the viral genome. Although a replication origin has not been defined for the poxviruses, cis-acting sequences found within the telomeric 200 bp have been implicated as necessary and sufficient for minichromosome replication. Replication occurs within cytoplasmic foci from approx 3 to 12 h postinfection. This chapter includes several methodologies to assay and quantitate replication in vivo, visualize replication foci microscopically, and test the integrity of central replication enzymes in vitro.

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC--C-3' in the presence of Mg2+ as shown generating 3' four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a betabetaalpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.

The Structure of Escherichia coli RusA Endonuclease Reveals a New Holliday Junction DNA Binding Fold

Holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in DNA recombination and repair. It is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the DNA, but their architecture varies. To date, with the exception of bacteriophage T4 endonuclease VII, each of the known resolvase enzyme structures has been categorized into one of two families: the integrases and the nucleases. We have now determined the structure of the Escherichia coli RusA Holliday junction resolvase, which reveals a fourth structural class for these enzymes. The structure suggests that dimer formation is essential for Mg 2+ cation binding and hence catalysis and that like the other resolvases, RusA distorts its Holliday junction target upon binding. Key residues identified by mutagenesis experiments are well positioned to interact with the DNA.

Fission yeast Mus81.Eme1 Holliday junction resolvase is required for meiotic crossing over but not for gene conversion.

Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81.Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81.Eme1 protein complex resolves Holliday junctions in meiotic cells.

Crystallization of RusA Holliday junction resolvase from Escherichia coli.

Crystals of the Escherichia coli Holliday junction resolvase RusA have been obtained using the hanging-drop method and characterized. The crystals have a primitive monoclinic form and belong to space group P2(1). The V(M) value suggests the presence of two copies of the monomer in the asymmetric unit. A full three-wavelength MAD data collection on a selenomethionine-incorporated form has been undertaken and structure determination is under way using data collected to 2.1 A resolution.

RNA interference inhibition of Mus81 reduces mitotic recombination in human cells.

Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex. In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function. Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo. We show that recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro. By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1. Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo. Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells. The recombination defect is rescued by expression of a bacterial Holliday junction resolvase. These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo.

Rapid analysis of protein–nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography

The RuvABC resolvasome of Escherichia coli typifies nucleoprotein complexes involved in genetic transactions. This molecular assembly catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have used matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) to rapidly identify the binding of RuvA to an immobilised synthetic Holliday junction; unambiguous identification was verified using tryptic digest of the bound protein. In conjunction with a novel fluorescent-based technique incorporating ion pair reverse phase liquid chromatography, a “footprint” of the RuvA:Holliday complex was obtained. These two complementary techniques offer a generic approach to the analysis of nucleoprotein complexes.

Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress.

Phagocytic cells inhibit the growth of intracellular pathogens by producing nitric oxide (NO). NO causes cell filamentation, induction of the SOS response, and DNA replication arrest in the Gram-negative bacterium Salmonella enterica. NO also induces double-stranded chromosomal breaks in replication-arrested Salmonella lacking a functional RecBCD exonuclease. This DNA damage depends on actions of additional DNA repair proteins, the RecG helicase, and RuvC endonuclease. Introduction of a recG mutation restores both resistance to NO and the ability of an attenuated recBC mutant Salmonella strain to cause lethal infection in mice, demonstrating that bacterial DNA replication is inhibited during host-pathogen interactions. Inhibition of DNA replication during nitrosative stress is invariably accompanied by zinc mobilization, implicating DNA-binding zinc metalloproteins as critical targets of NO-related antimicrobial activity.

Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance.

The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the alpha-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the truncated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif [Ceschini et al. (2001) EMBO J. 20, 6601-6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Deltaydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full-length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA 'phenotype'. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe.

Endonuclease cleavage of blocked replication forks: An indirect pathway of DNA damage from antitumor drug-topoisomerase complexes.

The cytotoxicity of several important antitumor drugs depends on formation of the covalent topoisomerase-DNA cleavage complex. However, cellular processes such as DNA replication are necessary to convert the cleavage complex into a cytotoxic lesion, but the molecular mechanism of this conversion and the precise nature of the cytotoxic lesion are unknown. Using a bacteriophage T4 model system, we have previously shown that antitumor drug-induced cleavage complexes block replication forks in vivo. In this report, we show that these blocked forks can be cleaved by T4 endonuclease VII to create overt DNA breaks. The accumulation of blocked forks increased in endonuclease VII-deficient infections, suggesting that endonuclease cleavage contributes to fork processing in vivo. Furthermore, purified endonuclease VII cleaved the blocked forks in vitro close to the branch points. These results suggest that an indirect pathway of branched-DNA cleavage contributes to the cytotoxicity of antitumor drugs that target DNA topoisomerases.