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1.  Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2 
PLoS ONE  2011;6(8):e23668.
Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C–80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly.
PMCID: PMC3157427  PMID: 21858199
2.  FAN1 Activity on Asymmetric Repair Intermediates Is Mediated by an Atypical Monomeric Virus-type Replication-Repair Nuclease Domain 
Cell Reports  2014;8(1):84-93.
FAN1 is a structure-selective DNA repair nuclease with 5′ flap endonuclease activity, involved in the repair of interstrand DNA crosslinks. It is the only eukaryotic protein with a virus-type replication-repair nuclease (“VRR-Nuc”) “module” that commonly occurs as a standalone domain in many bacteria and viruses. Crystal structures of three representatives show that they structurally resemble Holliday junction resolvases (HJRs), are dimeric in solution, and are able to cleave symmetric four-way junctions. In contrast, FAN1 orthologs are monomeric and cleave 5′ flap structures in vitro, but not Holliday junctions. Modeling of the VRR-Nuc domain of FAN1 reveals that it has an insertion, which packs against the dimerization interface observed in the structures of the viral/bacterial VRR-Nuc proteins. We propose that these additional structural elements in FAN1 prevent dimerization and bias specificity toward flap structures.
Graphical Abstract
•Bacterial proteins comprising solely a VRR-Nuc domain are dimeric in solution•Bacterial VRR-Nuc domains act as Holliday-junction-resolving enzymes•A conserved helical insertion in the FAN1 VRR-Nuc domain prevents dimerization•FAN1 monomers cannot cleave Holliday junctions and instead cleave 5′ flap DNA
The Fanconi anemia pathway is responsible for clearing DNA interstrand crosslinks that block DNA replication and transcription leading to genome instability. Here, Pennell et al. characterize the Fanconi-anemia-associated nuclease FAN1, comparing the structure and activity of its catalytic VRR-Nuc domain with prokaryotic examples. FAN1 is monomeric with 5′ flap specificity, whereas prokaryotic VRR-Nuc domains are dimeric Holliday-junction-resolving enzymes. FAN1 is proposed to contain a conserved helical insertion blocking dimer formation and consequently restricting substrate specificity.
PMCID: PMC4103454  PMID: 24981866
3.  DprB Facilitates Inter- and Intragenomic Recombination in Helicobacter pylori 
Journal of Bacteriology  2012;194(15):3891-3903.
For naturally competent microorganisms, such as Helicobacter pylori, the steps that permit recombination of exogenous DNA are not fully understood. Immediately downstream of an H. pylori gene (dprA) that facilitates high-frequency natural transformation is HP0334 (dprB), annotated to be a putative Holliday junction resolvase (HJR). We showed that the HP0334 (dprB) gene product facilitates high-frequency natural transformation. We determined the physiologic roles of DprB by genetic analyses. DprB controls in vitro growth, survival after exposure to UV or fluoroquinolones, and intragenomic recombination. dprB ruvC double deletion dramatically decreases both homologous and homeologous transformation and survival after exposure to DNA-damaging agents. Moreover, the DprB protein binds to synthetic Holliday junction structures rather than double-stranded or single-stranded DNA. These results demonstrate that the dprB product plays important roles affecting inter- and intragenomic recombination. We provide evidence that the two putative H. pylori HJRs (DprB and RuvC) have overlapping but distinct functions involving intergenomic (primarily DprB) and intragenomic (primarily RuvC) recombination.
PMCID: PMC3416544  PMID: 22609923
4.  A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems 
Nucleic Acids Research  2011;39(11):4532-4552.
The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins.
PMCID: PMC3113570  PMID: 21306995
5.  Crystal structure of RuvC resolvase in complex with Holliday junction substrate 
Nucleic Acids Research  2013;41(21):9945-9955.
The key intermediate in genetic recombination is the Holliday junction (HJ), a four-way DNA structure. At the end of recombination, HJs are cleaved by specific nucleases called resolvases. In Gram-negative bacteria, this cleavage is performed by RuvC, a dimeric endonuclease that belongs to the retroviral integrase superfamily. Here, we report the first crystal structure of RuvC in complex with a synthetic HJ solved at 3.75 Å resolution. The junction in the complex is in an unfolded 2-fold symmetrical conformation, in which the four arms point toward the vertices of a tetrahedron. The two scissile phosphates are located one nucleotide from the strand exchange point, and RuvC approaches them from the minor groove side. The key protein–DNA contacts observed in the structure were verified using a thiol-based site-specific cross-linking approach. Compared with known complex structures of the phage resolvases endonuclease I and endonuclease VII, the RuvC structure exhibits striking differences in the mode of substrate binding and location of the cleavage site.
PMCID: PMC3834835  PMID: 23980027
6.  Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases 
Journal of Bacteriology  2005;187(10):3431-3437.
Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.
PMCID: PMC1112027  PMID: 15866929
7.  Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution 
Nucleic Acids Research  2012;41(1):648-656.
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 ( RuvC). The crystal structure of RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but 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 RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of 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 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.
PMCID: PMC3592405  PMID: 23118486
8.  Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes 
Chromosoma  2011;120(2):109-127.
Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81–Mms4/EME1, Slx1–Slx4/BTBD12/MUS312, XPF–ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.
PMCID: PMC3057012  PMID: 21369956
9.  Comparative genomics of the FtsK–HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging 
Nucleic Acids Research  2004;32(17):5260-5279.
Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase. In this study, the FtsK–HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail. We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores. The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK–HerA superfamily. The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division. The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division. It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal–bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families. Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation. The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50. HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and, possibly, also during DNA replication. Extensive comparative analysis of the ‘genomic context’ combined with in-depth sequence analysis led to the prediction of numerous previously unnoticed nucleases of the NurA superfamily, including a specific version that is likely to be the endonuclease component of a novel restriction-modification system. This analysis also led to the identification of previously uncharacterized nucleases, such as a novel predicted nuclease of the Sir2-type Rossmann fold, and phosphatases of the HAD superfamily that are likely to function as partners of the FtsK–HerA superfamily ATPases.
PMCID: PMC521647  PMID: 15466593
10.  Two cDNAs from the plant Arabidopsis thaliana that partially restore recombination proficiency and DNA-damage resistance to E. coli mutants lacking recombination-intermediate-resolution activities. 
Nucleic Acids Research  1993;21(7):1647-1653.
Escherichia coli ruvC recG mutants lack RuvC endonuclease, which resolves crossed-strand joint molecules (Holliday junctions) formed during homologous recombination into recombinant products, and an activity (RecG) thought to partially replace RuvC. They are therefore highly deficient in homologous recombination, and sensitive to UV light and chemical DNA-damaging agents, presumably because of inability to tolerate unrepaired DNA damage by recombinational mechanisms (Lloyd, R.G. (1991) J. Bacteriol. 173:5414-5418). We transformed these mutants with plasmids expressing cDNAs from the plant Arabidopsis thaliana. Selection for bacteria with increased resistance to methylmethanesulfonate yielded two cDNAs, designated DRT111 and DRT112 (DNA-damage-repair/toleration). Expression of these plant cDNAs, especially DRT111, restored conjugal recombination proficiencies in ruvC and ruvC recG mutants to nearly wild-type levels. Both plant cDNAs significantly increased resistance of both mutants to UV light and several chemical DNA-damaging agents, but did not fully correct the mutant phenotypes. Drt111 activity, but not Drt112, also increased, to nearly wild-type levels, resistance of recG single mutants to UV plus mitomycin C. The predicted Drt111 and Drt112 polypeptides, 383 and 167 amino acids respectively, show no similarity with one another or with prokaryotic Holliday resolvases. Both appear chloroplast targeted; Drt112 is highly homologous to Arabidopsis plastocyanin. DRT111 and DRT112 probes hybridize only to DNA from closely related plants.
PMCID: PMC309376  PMID: 8479917
11.  The Enzyme and the cDNA Sequence of a Thermolabile and Double-Strand Specific DNase from Northern Shrimps (Pandalus borealis) 
PLoS ONE  2010;5(4):e10295.
We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies.
Methodology/Principal Findings
A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65°C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases.
The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.
PMCID: PMC2858651  PMID: 20421970
12.  Toxicity and tolerance mechanisms for azidothymidine, a replication gap-promoting agent, in Escherichia coli 
DNA repair  2010;10(3):260-270.
Azidothymidine (AZT, zidovudine) is used to treat HIV-AIDS and prevent maternal transmission to newborns. Because the azido group replaces the 3′ OH of thymidine, AZT is believed to act as a chain terminator during reverse transcription of viral RNA into DNA, although other mechanisms of viral inhibition have been suggested. There is evidence that AZT is genotoxic, particularly to the mitochondria. In this study, we use the bacterium Escherichia coli to investigate the mechanism of AZT toxicity and the cellular mechanisms that aid survival. We show that that replication arrests quickly after treatment with induction of the SOS DNA damage response. AZT appears to produce single-strand DNA gaps, as evident by RecF-dependent induction of the SOS response and visualization of single-strand DNA binding protein foci within the cell. Some of these gaps must be converted to breaks, since mutants in the RecBCD nuclease, required for recombinational double-strand break repair, are highly sensitive to AZT. Blocks in the late recombination functions, the RuvAB branch migration helicase and RuvC Holliday junction endonuclease, caused extreme AZT sensitivity that could be relieved by mutations in the early recombination functions, such as RecF, suggesting gaps engage in recombination reactions. Finally, our data suggest that the proofreading exonucleases of DNA polymerases play little role in AZT tolerance. Rather, Exonuclease III appears to be the enzyme that removes AZT: xthA mutants are highly AZT-sensitive, with a sustained SOS response, and overproduction of the enzyme protects wild-type cells. Our findings suggest that incorporation of AZT into human nuclear and mitochondrial DNA has the potential to promote genetic instability and toxicity through the production of ssDNA gaps and dsDNA breaks, and predicts that the human Exonuclease III ortholog, APE1, will be important for drug tolerance.
PMCID: PMC3046245  PMID: 21145792
gap repair; RecF pathway; recombination; SOS response
13.  Efficient Second Strand Cleavage during Holliday Junction Resolution by RuvC Requires Both Increased Junction Flexibility and an Exposed 5′ Phosphate 
PLoS ONE  2009;4(4):e5347.
Holliday junction (HJ) resolution is a critical step during homologous recombination. In Escherichia coli this job is performed by a member of the RNase H/Integrase superfamily called RuvC, whereas in Schizosaccharomyces pombe it has been attributed to the XPF family member Mus81-Eme1. HJ resolution is achieved through the sequential cleavage of two strands of like polarity at or close to the junction crossover point. RuvC functions as a dimer, whereas Mus81-Eme1 is thought to function as a dimer of heterodimers. However, in both cases the multimer contains two catalytic sites, which act independently and sequentially during the resolution reaction. To ensure that both strands are cleaved before the nuclease dissociates from the junction, the rate of second strand cleavage is greatly enhanced compared to that of the first. The enhancement of second strand cleavage has been attributed to the increased flexibility of the nicked HJ, which would facilitate rapid engagement of the second active site and scissile bond. Here we have investigated whether other properties of the nicked HJ are important for enhancing second strand cleavage.
Principal Findings
A comparison of the efficiency of cleavage of nicked HJs with and without a 5′ phosphate at the nick site shows that a 5′ phosphate is required for most of the enhancement of second strand cleavage by RuvC. In contrast Mus81-Eme1 cleaves nicked HJs with and without a 5′ phosphate with equal efficiency, albeit there are differences in cleavage site selection.
Our data show that efficient HJ resolution by RuvC depends on the 5′ phosphate revealed by incision of the first strand. This is a hitherto unappreciated factor in promoting accelerated second strand cleavage. However, a 5′ phosphate is not a universal requirement since efficient cleavage by Mus81-Eme1 appears to depend solely on the increased junction flexibility that is developed by the first incision.
PMCID: PMC2670506  PMID: 19399178
14.  Hjc resolvase is a distantly related member of the type II restriction endonuclease family 
Nucleic Acids Research  2000;28(22):4540-4543.
Hjc resolvase is an archaeal enzyme involved in homologous DNA recombination at the Holliday junction intermediate. However, the structure and the catalytic mechanism of the enzyme have not yet been identified. We performed database searching using the amino acid sequence of the enzyme from Pyrococcus furiosus as a query. We detected 59 amino acid sequences showing weak but significant sequence similarity to the Hjc resolvase. The detected sequences included DpnII, HaeII and Vsr endonuclease, which belong to the type II restriction endonuclease family. In addition, a highly conserved region was identified from a multiple alignment of the detected sequences, which was similar to an active site of the type II restriction endonucleases. We substituted three conserved amino acid residues in the highly conserved region of the Hjc resolvase with Ala residues. The amino acid replacements inactivated the enzyme. The experimental study, together with the results of the database searching, suggests that the Hjc resolvase is a distantly related member of the type II restriction endonuclease family. In addition, the results of our database searches suggested that the members of the RecB domain superfamily are evolutionarily related to the type II restriction endonuclease family.
PMCID: PMC113866  PMID: 11071943
15.  Structural, functional and evolutionary relationships between homing endonucleases and proteins from their host organisms 
Nucleic Acids Research  2012;40(12):5189-5200.
Homing endonucleases (HEs) are highly specific DNA-cleaving enzymes that are encoded by invasive DNA elements (usually mobile introns or inteins) within the genomes of phage, bacteria, archea, protista and eukaryotic organelles. Six unique structural HE families, that collectively span four distinct nuclease catalytic motifs, have been characterized to date. Members of each family display structural homology and functional relationships to a wide variety of proteins from various organisms. The biological functions of those proteins are highly disparate and include non-specific DNA-degradation enzymes, restriction endonucleases, DNA-repair enzymes, resolvases, intron splicing factors and transcription factors. These relationships suggest that modern day HEs share common ancestors with proteins involved in genome fidelity, maintenance and gene expression. This review summarizes the results of structural studies of HEs and corresponding proteins from host organisms that have illustrated the manner in which these factors are related.
PMCID: PMC3384342  PMID: 22406833
16.  Resolving branched DNA intermediates with structure-specific nucleases during replication in eukaryotes 
Chromosoma  2013;122(6):499-515.
Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81–Mms4/MUS81–EME1, Yen1/GEN1, and Slx1–Slx4/SLX1–SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication process.
PMCID: PMC3827899  PMID: 24008669
17.  Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family 
Nucleic Acids Research  2007;35(7):2377-2389.
Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.
PMCID: PMC1874659  PMID: 17392342
18.  Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements 
Biology Direct  2009;4:29.
In eukaryotes, RNA interference (RNAi) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown.
We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding) and PIWI (active or inactivated nuclease) domains, the prokaryotic Argonaute homologs (pAgos) fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ) pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases. Given these observations, the apparent extensive horizontal transfer of pAgo genes, and their common, statistically significant over-representation in genomic neighborhoods enriched in genes encoding proteins involved in the defense against phages and/or plasmids, we hypothesize that pAgos are key components of a novel class of defense systems. The PAZ-domain containing pAgos are predicted to directly destroy virus or plasmid nucleic acids via their nuclease activity, whereas the apparently inactivated, PAZ-lacking pAgos could be structural subunits of protein complexes that contain, as active moieties, the putative nucleases that we predict to be co-expressed with these pAgos. All these nucleases are predicted to be DNA endonucleases, so it seems most probable that the putative novel phage/plasmid-defense system targets phage DNA rather than mRNAs. Given that in eukaryotic RNAi systems, the PAZ domain binds a guide RNA and positions it on the complementary region of the target, we further speculate that pAgos function on a similar principle (the guide being either DNA or RNA), and that the uncharacterized domain found in putative operons with the short forms of pAgos is a functional substitute for the PAZ domain.
The hypothesis that pAgos are key components of a novel prokaryotic immune system that employs guide RNA or DNA molecules to degrade nucleic acids of invading mobile elements implies a functional analogy with the prokaryotic CASS and a direct evolutionary connection with eukaryotic RNAi. The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests.
This article was reviewed by Daniel Haft, Martijn Huynen, and Chris Ponting.
PMCID: PMC2743648  PMID: 19706170
19.  Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease. 
Journal of Bacteriology  1991;173(18):5747-5753.
The Escherichia coli ruvC gene is involved in DNA repair and recombination and encodes an endonuclease that resolves Holliday structure in vitro. The 2.8-kb chromosomal DNA fragment that encompasses the ruvC gene and its flanking regions was cloned and sequenced. Four open reading frames were identified in the order orf17-orf26-ruvC-orf23 immediately upstream of the ruvAB operon, and their orientations are the same as the ruvAB operon, except for orf23. Proteins encoded by orf17, orf26, and ruvC (orf19) were identified by the maxicell method, and their sizes agreed with those predicted from the DNA sequences. Among the open reading frames in this region, only ruvC is involved in the repair of UV-damaged DNA. ruvC appeared to be regulated by at least two promoters, but, in contrast to the ruvAB operon, ruvC is not regulated by the SOS system as demonstrated by operon fusions.
PMCID: PMC208306  PMID: 1885548
20.  Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily 
Nucleic Acids Research  2004;32(20):6129-6135.
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 ββα-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.
PMCID: PMC534630  PMID: 15562004
21.  Replication Fork Reversal after Replication–Transcription Collision 
PLoS Genetics  2012;8(4):e1002622.
Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.
Author Summary
Genomes are duplicated prior to cell division by DNA replication, and in all organisms replication impairment leads to chromosome instability. In bacteria, replication and transcription take place simultaneously, and in eukaryotes house-keeping genes are expressed during the S-phase; consequently, transcription is susceptible to impair replication progression. Here, we increase head-on replication–transcription collisions on the bacterial chromosome by inversion of a ribosomal operon (rrn). We show that only one recombination protein is required for growth when the rrn genes are highly expressed: the RecBCD complex, an exonuclease/recombinase that promotes degradation and RecA-dependent homologous recombination of linear DNA. In the absence of RecBCD, we observe linear DNA that ends in the collision region. This linear DNA is composed of only the origin-proximal region of the inverted rrn operon, indicating that it results from fork breakage. It is partly RuvABC-dependent (i.e. produced by the E. coli Holliday junction resolvase), indicating that blocked forks are reversed. The linear DNA ends up at the inverted rrn locus only if the RecJ exonuclease is inactivated; otherwise it is degraded, with major products ending in other upstream rrn operons, indicating that DNA degradation is slowed down by ribosomal operon sequences.
PMCID: PMC3320595  PMID: 22496668
22.  Identification of a single HNH active site in Type IIS restriction endonuclease Eco31I 
Journal of molecular biology  2007;370(1):157-169.
Type IIS restriction endonuclease Eco31I is a ‘short-distance cutter’, which cleaves DNA strands close to its recognition sequence, 5′-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I-PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in Type IIS restriction endonucleases that recognize common sequence core GTCTC.
PMCID: PMC2754561  PMID: 17499273
restriction endonuclease; Type IIS; HNH; endonuclease VII; active site
23.  Functional analysis of point mutations in human flap endonuclease-1 active site. 
Nucleic Acids Research  1997;25(16):3332-3338.
Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.
PMCID: PMC146887  PMID: 9241249
24.  Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site 
BMC Genomics  2005;6:21.
Prediction 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.
Using 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.
After 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.
PMCID: PMC551604  PMID: 15720711
25.  A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis 
Nucleic Acids Research  2002;30(2):482-496.
During a systematic analysis of conserved gene context in prokaryotic genomes, a previously undetected, complex, partially conserved neighborhood consisting of more than 20 genes was discovered in most Archaea (with the exception of Thermoplasma acidophilum and Halobacterium NRC-1) and some bacteria, including the hyperthermophiles Thermotoga maritima and Aquifex aeolicus. The gene composition and gene order in this neighborhood vary greatly between species, but all versions have a stable, conserved core that consists of five genes. One of the core genes encodes a predicted DNA helicase, often fused to a predicted HD-superfamily hydrolase, and another encodes a RecB family exonuclease; three core genes remain uncharacterized, but one of these might encode a nuclease of a new family. Two more genes that belong to this neighborhood and are present in most of the genomes in which the neighborhood was detected encode, respectively, a predicted HD-superfamily hydrolase (possibly a nuclease) of a distinct family and a predicted, novel DNA polymerase. Another characteristic feature of this neighborhood is the expansion of a superfamily of paralogous, uncharacterized proteins, which are encoded by at least 20–30% of the genes in the neighborhood. The functional features of the proteins encoded in this neighborhood suggest that they comprise a previously undetected DNA repair system, which, to our knowledge, is the first repair system largely specific for thermophiles to be identified. This hypothetical repair system might be functionally analogous to the bacterial–eukaryotic system of translesion, mutagenic repair whose central components are DNA polymerases of the UmuC-DinB-Rad30-Rev1 superfamily, which typically are missing in thermophiles.
PMCID: PMC99818  PMID: 11788711

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