Nicking Reaction Research Articles

The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3'-5' exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3'-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3' penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3'-5' exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3'-penultimate deoxyinosines derived from endo V nicking reaction.

Highly sensitive detection of microRNAs based on isothermal exponential amplification-assisted generation of catalytic G-quadruplexDNAzyme

It is well-known that microRNAs (miRNAs) have become an ideal class of biomarker candidates for clinical diagnosis of cancers, thus sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, as well as discovery of new targets for drugs. In this work, we have developed a sensitive method for microRNAs detection based on isothermal exponential amplification-assisted generation of catalytic G-quadruplex DNAzyme, and demonstrated its practical application in biological sample of cell lysate. The assay involves a combination of polymerase strand extension, single-strand nicking and catalytic reaction of G-quadruplex/hemin complex. It is designed such that, the target miRNA initiates the efficient synthesis of two kinds of short oligonucleotide fragments in the continuous cycle of the polymerization, nicking and displacement reactions, by means of thermostable polymerase and nicking endonuclease. One fragment has the same sequence as the target miRNA, except that the deoxyribonucleotides and thymine replace the ribonucleotides and uridine in the miRNA, to activate new cyclic chain reactions of polymerization, nicking and displacement reactions as the target miRNA. The other is the signal molecule of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. With such designed signal amplification processes, the proposed assay showed a quantitative analysis of sequence-specific miRNAs in a wide range from 1fM to 100nM with a low detection limit of 1fM. Moreover, this assay demonstrated excellent differentiation ability for the mismatch miRNAs targets and good performance in biological samples.

Kinetic Mechanism of Initiation by RepD as a Part of Asymmetric, Rolling Circle Plasmid Unwinding

Some bacterial plasmids carry antibiotic resistance genes and replicate by an asymmetric, rolling circle mechanism, in which replication of the two strands is not concurrent. Initiation of this replication occurs via an initiator protein that nicks one DNA strand at the double-stranded origin of replication. In this work, RepD protein from the staphylococcal plasmid pC221 carries this function and allows PcrA helicase to bind and begin unwinding the plasmid DNA. This work uses whole plasmid constructs as well as oligonucleotide-based mimics of parts of the origin to examine the initiation reaction. It investigates the phenomenon that nicking, although required to open a single-stranded region at the origin and so allow PcrA to bind, is not required for another function of RepD, namely to increase the processivity of PcrA, allowing it to unwind plasmid lengths of DNA. A kinetic mechanism of RepD initiation is presented, showing rapid binding of the origin DNA. The rate of nicking varies with the structure of the DNA but can occur with a rate constant of >25 s–1 at 30 °C. The equilibrium constant of the nicking reaction, which involves a transesterification to form a phosphotyrosine bond within the RepD active site, is close to unity.

Multiplex Loop-Mediated Isothermal Amplification Detection by Sequence-Based Barcodes Coupled with Nicking Endonuclease-Mediated Pyrosequencing

The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.

Divalent Metal Ion Differentially Regulates the Sequential Nicking Reactions of the GIY-YIG Homing Endonuclease I-BmoI

Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

Pyrosequencing on Nicked dsDNA Generated by Nicking Endonucleases

Although the pyrosequencing method is simple and fast, the step of ssDNA preparation increases the cost, labor, and cross-contamination risk. In this paper, we proposed a method enabling pyrosequencing directly on dsDNA digested by nicking endonucleases (NEases). Recognition sequence of NEases was introduced using artificially mismatched bases in a PCR primer (in the case of genotyping) or a reverse-transcription primer (in the case of gene expression analysis). PCR products were treated to remove excess amounts of primers, nucleotides, and pyrophosphate (PPi) prior to sequencing. After the nicking reaction, pyrosequencing starts at the nicked 3' end, and extension reaction occurs when the added dNTP is complementary to the non-nicked strand. Although the activity of strand displacement by Klenow is limited, approximately 10 bases are accurately sequenced; this length is long enough for genotyping and SRPP-based differential gene expression analysis. It was observed that the signals of two allele-specific bases in a pyrogram from nicked dsDNA are highly quantitative, enabling quantitative determination of allele-specific templates; thus, Down's Syndrome diagnosis as well as differential gene expression analysis was successfully executed. The results indicate that pyrosequencing using nicked dsDNA as templates is a simple, inexpensive, and reliable way in either quantitative genotyping or gene expression analysis.

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition.

DNA topoisomerases I and II (Top1 and Top2) are established molecular targets of anticancer drugs.1–5 Mammalian somatic cells express six topoisomerase genes: two TOP1 (TOP1 and TOP1mt), two TOP2 (TOP2α and β), two topoisomerase III (TOP3α and β)6,7 (Figure 1A). The most recently discovered eukaryotic topoisomerase is mitochondrial Top1 (Top1mt), which we reported in 2001.8,9 Figure 1 Schematic architecture of the topoisomerase cleavage complexes A common feature of topoisomerases is their catalytic mechanism, which in all cases consists in a nucleophilic attack of a DNA phosphodiester bond by a catalytic tyrosyl residue from the topoisomerase. The resulting covalent attachment of the tyrosine to the DNA phosphate is either at the 3′-end of the broken DNA in the case of Top1 enzymes (Top1 and Top1mt) or at the 5′-end of the broken DNA for the other topoisomerases (Figure 1). Thus, Top1 enzymes are the only topoisomerases that form a covalent link with the 3′-end of the broken DNA while generating a 5′-hydroxyl end at the other end of the break. In that respect, the eukaryotic Top1 enzymes belong to the broader family of site-specific tyrosine recombinases of prokaryotes and yeast (e.g., XerCD of Escherichia coli, bacteriophage λ integrase and Cre recombinase, and Flp of Saccharomyces cerevisiae). Another unique feature of the Top1 enzymes is their DNA relaxation mechanism by “controlled rotation” rather than by “strand passage”.10–12 In other words, Top1 enzymes relax DNA by letting the 5′-hydroxyl end swivel around the intact strand. This processive reaction does not require ATP or divalent metal binding, which is different from Top2 enzymes, which require both ATP hydrolysis and Mg2+.5,13 Top3 enzymes, which, like other type IA topoisomerases require Mg2+ (but no ATP) for catalysis14 are not very active in relaxing DNA supercoiling. They can relax DNA when it is very negatively supercoiled (single-stranded) one turn at a time.15 Moreover, both Top2 and Top3 enzymes change DNA topology by a strand passage distributive mechanism rather than by the processive controlled rotation of the Top1 enzymes. In the case of the Top2 enzymes, a full DNA duplex [referred to as the T (transported) strand] goes through the double-strand break made by an enzyme homodimer5,16,17 (Figure 1A). In the case of the Top3 enzymes, a single strand goes through the single-stranded break,14 typically at double-Holliday junction crossovers.18 The remarkable efficiency of the nicking-closing activity of Top1 enables the enzyme to relax both negatively and positively supercoiled DNA (even at 0°C)19 with similar efficiency.12 This is in contrast with Top2α, which relaxes more efficiently positive supercoiling.20 Of note, Top2β, like Top1 relaxes both positive and negative supercoils similarly.20 Removing positive supercoils is required for replication and transcription progression. Otherwise their accumulation in advance of replication and transcription complexes hinders the melting of the DNA duplex (by helicases) and consequently polymerase translocation along the DNA template. The normal nicking-closing activity of Top1 can however be uncoupled when the 5′-hydroxyl end generated by the nicking reaction becomes misaligned; for instance at preexisting base lesions or DNA nicks.21,22 In such cases, the Top1 cleavage complex (Top1cc) remains without effective legitimate religation partner. Those Top1-DNA covalent complexes are commonly referred to as “suicide complexes”. Under such conditions, Top1 can nevertheless religate an illegitimate (“foreign”) 5-hydroxyl-DNA end and act as a recombinase.23 This property is routinely used for molecular cloning (TOPO® Cloning, Invitrogen) using vaccinia Top1.24

TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate oriTs on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the oriT keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a "closed" conformation. In an IHF-deficient (IHF-) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF- background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short oriT loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an "open" form that exposes single-stranded DNA at the nic site, promoting the initial TrwC nicking reaction.

MutL-catalyzed ATP Hydrolysis Is Required at a Post-UvrD Loading Step in Methyl-directed Mismatch Repair

Methyl-directed mismatch repair is a coordinated process that ensures replication fidelity and genome integrity by resolving base pair mismatches and insertion/deletion loops. This post-replicative event involves the activities of several proteins, many of which appear to be regulated by MutL. MutL interacts with and modulates the activities of MutS, MutH, UvrD, and perhaps other proteins. The purified protein catalyzes a slow ATP hydrolysis reaction that is essential for its role in mismatch repair. However, the role of the ATP hydrolysis reaction is not understood. We have begun to address this issue using two point mutants: MutL-E29A, which binds nucleotide but does not catalyze ATP hydrolysis, and MutL-D58A, which does not bind nucleotide. As expected, both mutants failed to complement the loss of MutL in genetic assays. Purified MutL-E29A protein interacted with MutS and stimulated the MutH-catalyzed nicking reaction in a mismatch-dependent manner. Importantly, MutL-E29A stimulated the loading of UvrD on model substrates. In fact, stimulation of UvrD-catalyzed unwinding was more robust with MutL-E29A than the wild-type protein. MutL-D58A, on the other hand, did not interact with MutS, stimulate MutH-catalyzed nicking, or stimulate the loading of UvrD. We conclude that ATP-bound MutL is required for the incision steps associated with mismatch repair and that ATP hydrolysis by MutL is required for a step in the mismatch repair pathway subsequent to the loading of UvrD and may serve to regulate helicase loading.

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.

Reconstitution of a staphylococcal plasmid-protein relaxation complex in vitro.

The isolation of plasmid-protein relaxation complexes from bacteria is indicative of the plasmid nicking-closing equilibrium in vivo that serves to ready the plasmids for conjugal transfer. In pC221 and pC223, the components required for in vivo site- and strand-specific nicking at oriT are MobC and MobA. In order to investigate the minimal requirements for nicking in the absence of host-encoded factors, the reactions were reconstituted in vitro. Purified MobA and MobC, in the presence of Mg2+ or Mn2+, were found to nick at oriT with a concomitant phosphorylation-resistant modification at the 5' end of nic. The position of nic is consistent with that determined in vivo. MobA, MobC, and Mg2+ or Mn2+ therefore represent the minimal requirements for nicking activity. Cross-complementation analyses showed that the MobC proteins possess binding specificity for oriT DNA of either plasmid and are able to complement each other in the nicking reaction. Conversely, nicking by the MobA proteins is plasmid specific. This suggests the MobA proteins may encode the nicking specificity determinant.

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.

Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations

Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3′ hydroxyl and 5′ phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA.

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

Roles of TraI protein with activities of cleaving and rejoining the single-stranded DNA in both initiation and termination of conjugal DNA transfer.

The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood. We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region. Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.

Hexamminecobalt(III) chloride assisted, visible light induced, sequence dependent cleavage of DNA

9-Anthracene methyl ammonium chloride (AMAC) cleaves supercoiled pUC18 DNA with a marked preference for 3′-AG-5′ sites when the reaction mixture is irradiated with blue light (390 nm) in the presence of hexamminecobalt(III) chloride (CoHA). Irradiation of pUC18 DNA in the presence of either CoHA or AMAC alone, led to poor photocleavage, establishing a strong synergistic effect for the DNA nicking reaction. Irradiation of a mixture of AMAC and CoHA in the absence of DNA led to the photooxidation of AMAC to anthraquinone. Time resolved fluorescence studies show that AMAC fluorescence is quenched by DNA in a base sensitive manner. AMAC emission is quenched by CoHA at diffusion controlled rate, and the quenching events may involve electron transfer from AMAC to CoHA. The DNA may facilitate bimolecular electron transfer between the DNA-bound AMAC, and CoHA. The cation radical resulting from AMAC oxidation by CoHA is suggested to result in DNA cleavage. DNA sequencing experiments reveal preferential cleavage at 3′-AG-5′ sites. Among these cleavage sites, 3′-AGG-5′ sequences were associated with higher intensities. Only one out of 9 observed cleavage sites is not a 3′-AG-5′ sequence, instead it was a 3′-TGC-5′ site, containing a central G flanked by T/C instead of A/G. Little or no cleavage is observed at 3′-GCA-5′, or 3′-TGT-5′ sites. Several visible light absorbing agents bind to DNA at specific sites, and current observations may open the possibility of using these binding agents for sequence dependent photocleavage of DNA.

Initiation mechanisms in replication of filamentous phage DNA.

Filamentous phage DNA replication occurs in two steps. First, an RNA-primed minus strand is made on the plus strand. Then, a new plus strand is made on the resulting double strand. The RNA primer which initiates synthesis of the minus strand is produced at a specific site on the plus strand template by the host RNA polymerase holoenzyme. Despite a lack of sequences similar to the promoter consensus, the DNA replication origin has a much higher affinity for the holoenzyme than the transcriptional promoters. The non-template strand of the single-stranded -10 region of the origin appears to be responsible for this high affinity. The recognition mechanisms seem to share common features with those in transcriptional promoters. Plus-strand synthesis is initiated by a specific nick introduced into the negatively supercoiled replicative form by the phage-encoded initiator protein. The nicking reaction is preceded by an ordered series of protein-induced DNA conformational changes.

Genes involved in conjugative DNA processing of plasmid R6K.

The conjugative transfer region of the IncX plasmid R6K (TRA(x)) was analysed by transposon mutagenesis and DNA sequencing. Tn5tac1 insertional mutations localized TRA(x) to a 14.8 kb segment containing the alpha origin of transfer (oriT alpha), genes involved in conjugative DNA-processing (Dtr(x)) and genes involved in pilus synthesis and assembly (Mpf(x)). A second functional oriT, oriTbeta, was located at a distance of 5.3 kb from oriT alpha and was outside TRA(x). Mpf(x) occupied a segment of 10kb, as judged by the location of insertions conferring resistance to infection by the X pilus-specific phage X-2. At both sides of Mpf(x) there were insertions that were Tra but X-2 sensitive, suggesting that the mutations were in Dtr(x). This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriT alpha-taxA-taxC-Mpf(x)-taxB. taxC coded for a oriT-relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily. TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein pi, which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein pi for conjugation.