Analysis of substrate specificity of the RuvC holliday junction resolvase with synthetic Holliday junctions

The Escherichia coli RuvC protein endonucleolytically resolves Holliday junctions, which are formed as intermediates during genetic recombination and recombination repair. Previous studies using model Holliday junctions suggested that a certain size of central core of homology and a specific sequence in the junction were required for efficient cleavage by RuvC, although not for binding. To determine the minimum length of sequence homology required for RuvC cleavage, we made a series of synthetic Holliday junctions with various lengths of homologous sequence in the core region. It was demonstrated that a monomobile junction possessing only 2 base pairs of the homology core was efficiently cleaved by RuvC. To study the sequence specificity for cleavage, we made 16 bimobile junctions, which differed only in the homologous core sequence. Among them, 6 junctions were efficiently cleaved. Cleavage occurred by introduction of nicks symmetrically at the 3'-side of thymine in all cases. However, the nucleotide bases at the 3'-side of the thymines were not always identical between the two strands nicked. These results suggest that RuvC recognizes mainly topological symmetry of the Holliday junction but not the sequence symmetry per se, that the thymine residue at the cleavage site plays an important role for RuvC-mediated resolution, and that a long homologous core sequence is not essential for cleavage.     

Sequence specificity and biochemical characterization of the RusA Holliday junction resolvase of Escherichia coli

The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5' of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses approximately 80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+ strains unless it is expressed at a high level.     

Recognition and manipulation of branched DNA by the RusA Holliday junction resolvase of Escherichia coli

Homologous recombination is a fundamental cellular process that shapes and reshapes the genomes of all organisms and promotes repair of damaged DNA. A key step in this process is the resolution of Holliday junctions formed by homologous DNA pairing and strand exchange. In Escherichia coli , a Holliday junction is processed into recombinant products by the concerted activities of the RuvA and RuvB proteins, which together drive branch migration, and RuvC endonuclease, which resolves the structure. In the absence of RuvABC, recombination can be promoted by increasing the expression of the RusA endonuclease, a Holliday junction resolvase encoded by a cryptic prophage gene. Here, we describe the DNA binding properties of RusA. We found that RusA was highly selective for branched molecules and formed complexes with these structures even in the presence of a large excess of linear duplex DNA. However, it does bind weakly to linear duplex DNA. Under conditions where there was no detectable binding to duplex DNA, RusA formed a highly structured complex with a synthetic Holliday junction that was remarkably stable and insensitive to divalent metal ions. The duplex arms were found to adopt a specific alignment within this complex that approximated to a tetrahedral conformation of the junction.     

Substrate specificity of the Escherichia coli RuvC protein. Resolution of three- and four-stranded recombination intermediates

The specificity of the Escherichia coli RuvC Holliday junction resolvase has been investigated in vitro. RuvC protein cleaves synthetic DNA substrates that model three- or four-stranded recombination intermediates but fails to act upon Y junctions, G/A mismatches, heterologous loop structures, or two-stranded branched junctions. RuvC therefore differs from endonuclease VII of bacteriophage T4 which exhibits broad range specificity. Using related three- and four-stranded synthetic DNA junctions, we show that RuvC cleaves both junctions at the same DNA sequence and requires a region of homology at the junction point. The action of RuvC on three- and four-stranded recombination intermediates made by RecA was also investigated. We found that RuvC fails to resolve three-stranded intermediates in the presence of RecA, although four-stranded intermediates are resolved under the same conditions. However, both three- and four-stranded intermediates are substrates for the nuclease after removal of RecA. We interpret these differences in terms of the contiguity of the RecA nucleoprotein filament which may, under certain conditions, limit access to the Holliday junction resolvase.     

Random walk models for DNA synapsis by resolvase

During site-specific recombination by resolvase, the protein binds to two sites on a supercoiled DNA molecule and the loaded sites then interact with each other to form a synaptic complex. The kinetics of synapsis show non-exponential behaviour extending over five log units of time and are independent of the length of the DNA molecule and the length of DNA between the sites. In this study, numerical models were developed in order to account for how fluctuations in the structure of supercoiled DNA might lead to the juxtaposition of distant sites in a manner consistent with the experimental data on synapsis by resolvase. Models where the juxtaposition arises from fluctuations around branch points in the superhelix failed to match the data: they yielded non-exponential kinetics but only over two log units of time and they predicted longer synapsis times for both larger DNA molecules and larger inter-site spacings. In another model, one fraction of the juxtaposition events gives rise directly to the productive complex while the remaining fraction initially yields a non-productive complex: the latter molecules undergo no further fluctuations until the abortive synapse dissociates at the end of a delay period. This model again failed to match the experimental data. However, the inclusion of three sorts of non-productive complexes, each with a different delay constant, led to progress curves that concurred with the data. Schemes were also developed to account for the juxtaposition of three sites at a branch point in supercoiled DNA.     

Intermolecular cleavage by UmuD-like enzymes: identification of residues required for cleavage and substrate specificity

The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis. An essential step in this process is the enzymatic self-processing of the UmuD-like proteins. This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism. Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD (UmuDSt) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuDSt enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuDSt. We have further localized the residue predominantly responsible for UmuDSt-catalyzed heterologous cleavage to Ser50 in the substrate molecule. We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuDSt enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observation that mutations known to disrupt an E. coli UmuD' filament dimer also block intermolecular UmuDEc cleavage.     

Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase

Binding of a cleavage-incompetent mutant of the Flp recombinase induces a roughly square-planar geometry in synthetic immobile Holliday junctions. The branch points, which are rigidly fixed in these junctions in their free forms, tend to be more flexible in their protein-bound forms. Our results (1) suggest a plausible mechanism for the switching of the recombination complex from the Holliday-forming mode to the Holliday-resolving mode, (2) provide a rationale for previous observations that Flp resolves preformed immobile Holliday structures in the parental or in the recombinant mode in a relatively unbiased manner, and (3) accommodate two modes of DNA cleavage by Flp (transhorizontal or transdiagonal) in Holliday substrates.     

Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease

The SfiI endonuclease differs from other type II restriction enzymes by cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The nature of this communication event between distant DNA sites was analysed on plasmids with recognition sites for SfiI interspersed with recombination sites for resolvase. These were converted by resolvase to catenanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions between SfiI sites on the same DNA therefore cannot follow the DNA contour and, instead, must stem from their physical proximity. In buffer lacking Mg2+, where SfiI is inactive while resolvase is active, the addition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging its sites and thus isolating the sites for resolvase into separate loops. The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+.     

Substrate specificity of delta ribozyme cleavage

The specificity of delta ribozyme cleavage was investigated using a trans-acting antigenomic delta ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min-1, an apparent Km of 17.9 nM and an apparent second-order rate constant of 1.89 x 10(7) min-1 M-1. The substrate specificity of the delta ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of delta ribozyme cleavage.     

Substrate specificity of the ultraviolet-endonuclease from Micrococcus luteus. Endonucleolytic cleavage of depurinated DNA

The ultraviolet-endonuclease isolated from Micrococcul luteus, specific for pyrimidine dimers, is able to attack not only ultraviolet-irradiated DNA (leading to 3'OH-5'PO4 single-strand breaks) but also superhelical covalently-closed circular DNA of phage lambda damaged by heating at 70 degrees C, pH 5.93. The number of endonuclease-sensitive defects in the DNA corresponds to the number of alkalilabile bonds (apurinic sites) induced by heating. Competition between ultraviolet-induced lesions and apurinic sites for ultraviolet-endonuclease is demonstrated; the affinity of the enzyme for pyrimidine dimers is about three times that for apurinic sites. Both activities of the ultraviolet-endonuclease are inactivated at 50 degrees C at the same rate. The ultraviolet-endonuclease is able to reduce the infectious activity of depurinated lambda DNA towards Ca2+-treated uvr+ and uvr A Escherichia coli cells. It is concluded that both pyrimidine dimers and apurinic sites can be recognized by one and the same enzyme (the ultraviolet-endonuclease).     

Induction of mammalian DNA topoisomerase I-mediated DNA cleavage and DNA winding by bulgarein

Bulgarein, a fungal metabolite, induced mammalian topoisomerase I-mediated DNA cleavage in vitro. The cleavage activity of bulgarein was comparable to that of camptothecin at a drug concentration range of 0.025-approximately 5 microM. The DNA cleavage induced by bulgarein was suppressed at concentrations above 12.5 microM. Treatment of a reaction mixture containing bulgarein and topoisomerase I with elevated temperature (65 degrees C) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I-mediated DNA cleavage induced by bulgarein is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex." Intensity of cleaved DNA fragments induced by bulgarein with topoisomerase I was different from that induced by camptothecin. Bulgarein inhibited catalytic activities of both topoisomerase I and topoisomerase II. The changes in absorption spectra of bulgarein in the visible region observed upon addition of increasing amounts of calf thymus DNA indicate that bulgarein interacts with DNA. DNA (un)winding assay by two-dimensional gel electrophoresis showed that bulgarein induced the winding of DNA in the opposite direction to that of an intercalator so that positively supercoiled molecules are produced. Thus, bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist.     

Dissection of the sequence specificity of the Holliday junction endonuclease CCE1

CCE1 is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomycescerevisiae. The junction-resolving enzymes are a diverse class, widely distributed in nature from viruses to higher eukaryotes. In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent. We have undertaken a systematic study of the sequence specificity of CCE1, using a single-turnover kinetic assay and a panel of synthetic four-way DNA junction substrates. A tetranucleotide consensus cleavage sequence 5'-ACT downward arrowA has been identified, with specificity residing mainly at the central CT dinucleotide. Equilibrium constants for CCE1 binding to four-way junctions are unaffected by sequence variations, suggesting that substrate discrimination occurs predominantly in the transition state complex. CCE1 cuts most efficiently at the junction center, but can also cleave the DNA backbone at positions one nucleotide 3' or 5' of the point of strand exchange, suggesting a significant degree of conformational flexibility in the CCE1:junction complex. Introduction of base analogues at single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail. In particular, the N7 moiety of the guanine base-pairing with the cytosine of the consensus sequence appears to be crucial for catalysis. The functional significance of sequence specificity in junction-resolving enzymes is discussed.     

Sequence dependence and direct measurement of crossover isomer distribution in model Holliday junctions using NMR spectroscopy

A 32-base-pair model of the Holliday junction (HJ) intermediate in genetic recombination has been prepared and analyzed in-depth by 2D and 3D (1)H NMR spectroscopy. This HJ (J2P1) corresponds to a cyclic permutation of the base pairs at the junction relative to a previously studied HJ [J2; Chen, S.-M., & Chazin, W.J. (1994) Biochemistry 33, 11453-11459], designed to probe the effect of the sequence at the n - 1 position (where n is the residue directly at the branch point) on the stacking geometry. Observation of several interbase nuclear Overhauser effects (NOEs) clearly indicates a strong preference for the isomer opposite that observed for J2, confirming the dependence of stacking isomer preference on the sequence at the junction. As for other model HJs studied, a small equilibrium distribution of the alternate isomer could be identified. A sample of J2P1 was prepared with a single (15)N-labeled thymine residue at the branch point. 1D (15)n-filtered (1)H-detected experiments on this sample at low temperature give strong support for the co-existence of the two stacking isomers and provide a much more direct and accurate measure of the crossover isomer distribution. The comparative analysis of our immobile HJs and a model cruciform structure [Pikkemaat, J.A., van den Elst, H., van Boom, J.H., & Altona, C. (1994) Biochemistry 33, 14896-14907] sheds new light on the issue of the relevance of crossover isomer preference in vivo.     

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.     

Mechanism of double-strand DNA cleavage effected by iron-bleomycin

We have observed that in the absence of hydrogen peroxide the Fe(III)-bleomycin (BLM) complex exhibits high DNA cleavage efficiency, converting supercoiled Form I DNA (pBR322 or phix174) to Form II (nicked, relaxed circular); the present study may give an important clue to elucidate the fact that iron-bleomycin mediated double-strand DNA cleavage requires at least one molecule of oxygen (O2) over the amount required to form 'activated bleomycin."     

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.     

Three-way and four-way junctions in DNA: a conformational viewpoint

DNA junctions are potential intermediates in various important genetic processes, including mutagenesis and recombination. The quantity of research carried out in this area is rapidly increasing. Examples of three-way and four-way junctions are now relatively well characterized and a few common properties have been recognized, of which the most important is the tendency of junctions to fold into one or more coaxially stacked helical conformations or cross-over structures.     

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.     

DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI

Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns. They have also been identified in self-splicing inteins (protein introns). These enzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-specific double-strand break at a target site in an allele that lacks the corresponding mobile intron. The target sites recognized by these small endonucleases are generally long (14-44 base pairs). Four families of homing endonucleases have been identified, including the LAGLIDADG, the His-Cys box, the GIY-YIG and the H-N-H endonucleases. The first identified His-Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 A resolution. I-PpoI displays an elongated fold of dimensions 25 x 35 x 80 A, with mixed alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long alpha-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 A apart. The enzyme possesses a new zinc-bound fold and endonuclease active site. The structure has been determined in both uncleaved substrate and cleaved product complexes.     

Ribonuclease P substrate specificity: cleavage of a bacteriophage phi80-induced RNA

RNase P can cleave in vitro a bacteriophage phi80-induced RNA which is 62 nucleotides long [M3 RNA, G. Pieczenik et al. (1972) Arch. Biochem. Biophys. 152, 152-165] to yield two specific fragments 25 and 37 nucleotides long. As is the case for another substrate of RNase P; the precursor to Escherichia coli 4.5S RNA, the cleavage site in M3 RNA is at the end of a long double-stranded region immediately adjacent to a single-stranded segment. Similar nucleotide sequences span the cleavage site in both substrates. These and other features of the reaction of RNase P with M3 and 4.5S precursor RNA are different from some aspects of the reaction of this enzyme with tRNA precursor molecules. A qualitative scheme is presented that is directed towards the understanding of the differences in RNase P cleavage site specificity for these substrates.