Single-strand Nicks Research Articles

Chromosomal Integration Mechanism of Infecting Mu Virion DNA

DNA transposition is central to the propagation of temperate phage Mu. A long-standing problem in Mu biology has been the mechanism by which the linear genome of an infecting phage, which is linked at both ends to DNA acquired from a previous host, integrates into the new host chromosome. If Mu were to use its well-established cointegrate mechanism for integration (single-strand nicks at Mu ends, joined to a staggered double-strand break in the target), the flanking host sequences would remain linked to Mu; target-primed replication of the linear integrant would subsequently break the chromosome. The absence of evidence for chromosome breaks has led to speculation that infecting Mu might use a cut-and-paste mechanism, whereby Mu DNA is cut away from the flanking sequences prior to integration. In this study we have followed the fate of the flanking DNA during the time course of Mu infection. We have found that these sequences are still attached to Mu upon integration and that they disappear soon after. The data rule out a cut-and-paste mechanism and suggest that infecting Mu integrates to generate simple insertions by a variation of its established cointegrate mechanism in which, instead of a "nick, join, and replicate" pathway, it follows a "nick, join, and process" pathway. The results show similarities with human immunodeficiency virus integration and provide a unifying mechanism for development of Mu along either the lysogenic or lytic pathway.

Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination

Mismatch repair (MMR) proteins are important for antibody class-switch recombination (CSR), but their roles are unknown. We propose a model for the function of MMR in CSR in which MMR proteins convert single-strand nicks instigated by activation-induced cytidine deaminase (AID) into the double-strand breaks (DSBs) that are required for CSR. This model does not invoke any novel functions for MMR but simply posits that, owing to numerous single-strand nicks in the switch (S) regions of both DNA strands, when MMR proteins are recruited by U:G mismatches, they excise one strand of DNA and soon reach a nick on the opposite strand. This halts excision activity and creates a DSB. This model explains why B cells that lack either S mu and MSH2 or UNG and MSH2 cannot undergo CSR.

Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis

Variable (diversity) joining (V(D)J) recombination is initiated by the introduction of single-strand DNA breaks (nicks) at recombination signal sequences (RSSs). The importance and fate of these RSS nicks for the regulation of the V(D)J rearrangement and their potential contribution to genomic instability are poorly understood. Using two new methodologies, we were able to detect and quantify specific RSS nicks introduced into genomic DNA by incubation with recombination-activating gene proteins in vitro. In vivo, however, we found that nicks mediated by recombination-activating gene (RAG) proteins were detectable only in gene segments associated with RSSs containing 12-base pair spacers but not in those containing 23-base pair spacers. These data support a model of capture rather than synapsis for pairwise RSS cleavage during V(D)J recombination.

Roles of Double-strand Breaks, Nicks, and Gaps in Stimulating Deletions of CTG·CAG Repeats by Intramolecular DNA Repair

A series of plasmids harboring CTG.CAG repeats with double-strand breaks (DSB), single-strand nicks, or single-strand gaps (15 or 30 nucleotides) within the repeat regions were used to determine their capacity to induce genetic instabilities. These plasmids were introduced into Escherichia coli in the presence of a second plasmid containing a sequence that could support homologous recombination repair between the two plasmids. The transfer of a point mutation from the second to the first plasmid was used to monitor homologous recombination (gene conversion). Only DSBs increased the overall genetic instability. This instability took place by intramolecular repair, which was not dependent on RuvA. Double-strand break-induced instabilities were partially stabilized by a mutation in recF. Gaps of 30 nt formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nt did not induce expansions or deletions. Formation of this deletion product required the CTG.CAG repeats to be present in the single-stranded region and was stimulated by E.coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the intramolecular repair-induced instabilities and the formation of the 30 nt deletion product.

Structures of Three Classes of Anticancer Agents Bound to the Human Topoisomerase I−DNA Covalent Complex

Human topoisomerase I (top1) is the molecular target of a diverse set of anticancer compounds, including the camptothecins, indolocarbazoles, and indenoisoquinolines. These compounds bind to a transient top1-DNA covalent complex and inhibit the resealing of a single-strand nick that the enzyme creates to relieve superhelical tension in duplex DNA. (Hertzberg, R. P.; et al. Biochem. 1989, 28, 4629-4638. Hsiang, Y. H.; et al. J. Biol. Chem 1985, 260, 14873-14878. Champoux, J. J. Annu. Rev. Biochem. 2001, 70, 369-413. Stewart, L.; et al. Science 1998, 729, 1534-1541.) We report the X-ray crystal structures of the human top1-DNA complex bound with camptothecin and representative members of the indenoisoquinoline and indolocarbazole classes of top1 poisons. The planar nature of all three structurally diverse classes allows them to intercalate between DNA base pairs at the site of single-strand cleavage. All three classes of compounds have a free electron pair near Arg364, a residue that if mutated confers resistance to all three classes of drugs. The common intercalative binding mode is augmented by unexpected chemotype-specific contacts with amino acid residues Asn352 and Glu356, which adopt alternative side-chain conformations to accommodate the bound compounds. These new X-ray structures explain how very different molecules can stabilize top1-DNA covalent complexes and will aid the rational design of completely novel structural classes of anticancer drugs.

Characterization of a Cloned Temperature-Sensitive Construct of the Diphtheria Toxin A Domain

Our goal was to determine how the DTM mutant construct of the A domain of diphtheria toxin (DTx) causes temperature-sensitive effects in Drosophila and yeast [Bellen, H. J., D'Evelyn, D., Harvey, M., Elledge, S. J. (1992) Development 114, 787-796]. Because DTM fortuitously bears the same point mutation as found in the A chain of CRM197, an ADP-ribosyltransferase (ADPrT)-deficient form of DTx, we hypothesized that the dramatic low-temperature-sensitive effects did not stem from ADP-ribosylation of elongation factor 2 (EF-2). To rule out acquisition of ADPrT activity at low temperatures, we assayed mutant forms of the A domain of DTx produced by in vitro transcription/translation and found that DTM has no ability to ADP-ribosylate EF-2 at 18 or 30 degrees C. Because the DTM gene results in a protein with a 23-amino acid missense carboxy-terminal extension, we also constructed a form without this extension. Assays for nuclease activity revealed that nuclease activity comigrated with the two distinguishable E. coli-cloned mutant proteins DTM and DTM-23, regardless of whether electrophoresis was conducted under denaturing or nondenaturing conditions in gels embedded with DNA. Studies with CRM197 showed that Ca(2+) and Mg(2+) promote single-strand DNA nicks, whereas Mn(2+) promotes double-strand DNA breaks. Evidence that the cation-dependent nuclease and NAD-dependent ADPrT enzymic sites are distinct is that NAD protected only the A domain of DTx from proteolytic cleavage, whereas DNA protected the A domains of both DTx and CRM197. We conclude that the nuclease activity of DTM is responsible for the temperature-sensitive effects associated with its expression in both yeast and Drosophila.

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.

Hydroxyl Radical is the Active Species in Photochemical DNA Strand Scission by Bis(peroxo)vanadium(V) Phenanthroline

Bis(peroxo)vanadium(V) complexes are widely investigated as anticancer agents. They exert their antitumor and cyctotoxic effects through inhibition of tyrosine phosphatases and DNA cleavage, respectively. The latter process remains poorly understood. The mechanism of DNA cleavage by NH(4)[(phen)V(O)(eta(2)-O(2))(2)] (phen = 1,10-phenanthroline) was investigated. Kinetic studies on DNA cleavage revealed that the complex is a single-strand nicking agent with no specificity. EPR experiments using 2,2,6,6-tetramethyl-4-piperidone (TMP) and 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) as spin-traps for singlet oxygen and hydroxyl radical, respectively, implicated hydroxyl radical production upon photodecomposition of bis(peroxo)vanadium(V). This was corroborated by benzoate inhibition of DNA strand scission and stoichiometric oxidation of 2-propanol to acetone upon irradiation of bis(peroxo)vanadium(V) phenanthroline. High-resolution polyacrylamide gel analysis of the vanadium cleavage reaction and [Fe(II)EDTA](2)(-)/H(2)O(2) resulted in comigration of "ladder" pattern bands, which superimposed when both reactions were run on the same lane. These findings identify hydroxyl radical produced from the photooxidation of the peroxo ligand on vanadium as the active species in DNA cleavage.

Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin

Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks. Circular single-stranded DNA (ssDNA) is also a substrate for endonucleolytic attack. Pre-incubation of DTx with a 2000-fold excess of NAD, the natural substrate for the toxin's ADP-ribosyltransferase (ADPrT) activity, inhibited the transfer of radiolabeled ADP-ribose to elongation factor 2 but had no effect on the degradation of radiolabeled DNA. Based on this result and the fact that compounds known to inhibit the ADPrT activity of DTx had no effect on its nuclease activity and pre-incubation of DTx with DNA had no effect on ADPrT activity, we conclude that the ADPrT and nuclease active sites of DTx are functionally and spatially distinct. Moreover, studies with an ADPrT-inactivated form of DTx indicate that nuclease activity alone can lead to target cell lysis.

Solution Structure and DNA Binding of the Zinc-finger Domain from DNA Ligase IIIα

DNA ligase IIIα carries out the final ligation step in the base excision repair (BER) and single strand break repair (SSBR) mechanisms of DNA repair. The enzyme recognises single-strand nicks and other damage features in double-stranded DNA, both through the catalytic domain and an N-terminal domain containing a single zinc finger. The latter is homologous to other zinc fingers that recognise damaged DNA, two in the N terminus of poly(adenosine-ribose)polymerase and three in the N terminus of the Arabidopsis thaliana nick-sensing DNA 3′-phosphoesterase. Here, we present the solution structure of the zinc-finger domain of human DNA ligase IIIα, the first structure of a finger from this group. It is related to that of the erythroid transcription factor GATA-1, but has an additional N-terminal β-strand and C-terminal α-helix. Chemical shift mapping using a DNA ligand containing a single-stranded break showed that the DNA-binding surface of the DNA-ligase IIIα zinc finger is substantially different from that of GATA-1, consistent with the fact that the two proteins recognise very different features in the DNA. Likely implications for DNA binding are discussed.

Bacteriophage T4 endonuclease II: concerted single-strand nicks yield double-strand cleavage.

In vivo, endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated. In vitro, however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks. Upper strand nick positions vary both in vivo and in vitro. A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII. Using DNA ligase in vitro, we found that EndoII nicked both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site. This indicates that the right-side CSE at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage. Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand. In addition, the upper and lower strands were nicked independently of each other, seldom resulting in double-strand cleavage. Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.

Genesis of the strand‐biased signature in somatic hypermutation of rearranged immunoglobulin variable genes

The history and current development of the reverse transcriptase model of somatic hypermutation (RT-model) is reviewed with particular reference to the genesis of strand-biased mutation signatures in rearranged immunoglobulin variable genes (V(D)J). The recent disagreement in the field as to whether strand bias really exists or not has been critically analysed and the confusion traced to the putative presence, in some mutated V(D)J sequence collections, of polymerase chain reaction (PCR)-recombinant artefacts. Recent analysis of somatic hypermutation in xeroderma pigmentosum variant patients, by the group of PJ Gearhart and others, has established that the Y-family translesion DNA repair enzyme, DNA polymerase eta (eta), is responsible for the striking A-T targeted strand-bias mutation signature seen in all mouse and human collections of somatically mutated V(D)J sequences. This evidence, together with our own recent demonstration that human DNA polymerase eta is a reverse transcriptase, leads to the conclusion that the strand-biased A-T mutation signature is caused either by: (i) error-prone DNA-dependent DNA repair synthesis by pol-eta of single-strand nicks preferentially in the non-transcribed strand; and/or (ii) by error-prone cDNA synthesis of the transcribed strand by pol-eta using the pre-mRNA as the copying template, primed by the nicked transcribed DNA strand, followed by replacement of the original transcribed strand by cDNA. Analysis of the total mutation pattern also suggests that the major transitions observed in SHM (A-->G, C-->T and G-->A) can be explained by known RNA editing mechanisms active on pre-mRNA which are then written into cDNA during synthesis of the transcribed strand by error-prone cellular reverse transcriptases such as pol-eta.

Identification and characterization of a conserved nuclease secreted by strains of the Lactobacillus casei group.

Nuclease secretion was evaluated for five species of Lactobacillus and the activity was characterized in terms of thermal resistance, molecular weight and mode of action on plasmid DNA. Assays of nuclease from L. rhamnosus ATCC 9595 on DNA of different origins indicates a broad activity spectrum. Secreted nuclease from this strain resists a thermal treatment of 20 min at 100 degrees C, is not sensitive to a treatment for disruption of disulphide bonds nor to EDTA treatment under 10 mM l(-1). Nuclease production is not growth linked and seems to be constitutive. Extracellular nuclease of L. rhamnosus ATCC 9595 introduces a single-stranded nick in supercoiled DNA, thus potentially reducing the transformability of plasmid DNA. In seven of eight tested strains, SDS-PAGE revealed a major protein with a molecular weight of ca 35 kDa. Minor degradation products also showed nuclease activity. A comparative analysis of the extracellular fractions of 14 different Lactobacillus strains indicate that nuclease secretion seems to be a widely distributed function among species of milk-related lactobacilli. The production of secreted nuclease may contribute to the low ability of Lactobacillus spp. to be transformed and maintain exogenous DNA. Determination of the characteristics and distribution of nuclease activity contribute to developing strategies to overcome this barrier to efficient transformation of milk lactobacilli.

Cleavage of double-stranded DNA by the intrinsic 3′-5′ exonuclease activity of DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus at high temperature

The substrate requirement of the intrinsic 3′-5′ exonuclease of DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus P2 ( Sso polB1) was investigated. Sso polB1 degraded both single-stranded (ss) and double-stranded (ds) DNA at similar rates in vitro at temperatures of physiological relevance. No difference was found in the cleavage of 3′-recessive, 3′-protruding and blunt-ended DNA duplexes at these temperatures. However, a single-stranded nick in duplex DNA was less readily employed by the enzyme to initiate cleavage than a free 3′ end. At lower temperatures, Sso polB1 cleaved ssDNA more efficiently than dsDNA. The strong 3′-5′ exonuclease activity of polB1 was inhibited by 50% in the presence of 2 μM dNTPs, but remained measurable at up to 600 μM dNTPs. In view of the strong exonuclease activity of Sso polB1 on matched dsDNA, we suggest that S. solfataricus may have evolved mechanisms to regulate the exonuclease/polymerase ratio of the enzyme, thereby reducing the cost of proofreading at high temperature.

Novel naphthalimide hydroperoxide photonucleases: The role of thiocyclic-Fused area and the difference in spectra, photochemistry and photobiological activity

Novel five- and six-membered thiocyclic-fused naphthalimide hydroperoxides ( 7, 8) as photonucleases were designed, synthesized via unusual isomerization in Pschorr cyclization and photooxygenation. The five-membered 7 was able to induce single-strand nicks in duplex DNA pH independently (7.0–8.5) at 0.5 μM under 366 nm, while the six-membered 8 could photonick the duplex DNA pH dependently (7.0–8.5) at 5 μM under 450 nm and showed ‘time-controlled’ photo-bioactivity. Their thiocycles and the angular conjugated plane have contributions to their binding affinity with DNA and photocleaving efficiency.

Resolution of parvovirus dimer junctions proceeds through a novel heterocruciform intermediate.

The minute virus of mice initiator protein, NS1, excises new copies of the left viral telomere in a single sequence orientation, dubbed flip, during resolution of the junction between monomer genomes in palindromic dimer intermediate duplexes. We examined this reaction in vitro using both (32)P-end-labeled linear substrates and similar unlabeled templates labeled by incorporation of [alpha-(32)P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriL(TC), the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3' nucleotide to prime DNA synthesis and, in a reaction we call "read-through synthesis," forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5' end of the DNA, where it can serve as a 3'-to-5' helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriL(GAA).

Cell death induced by granzyme C

AbstractAlthough the functions of granzymes A and B have been defined, the functions of the other highly expressed granzymes (Gzms) of murine cytotoxic lymphocytes (C, D, and F) have not yet been evaluated. In this report, we describe the ability of murine GzmC (which is most closely related to human granzyme H) to cause cell death. The induction of death requires its protease activity and is characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. The kinetics of these events are similar to those caused by granzyme B, and its potency (defined on a molar basis) is also equivalent. The induction of death did not involve the activation of caspases, the cleavage of BID, or the activation of the CAD nuclease. However, granzyme C did cause rapid mitochondrial swelling and depolarization in intact cells or in isolated mitochondria, and this mitochondrial damage was not prevented by cyclosporin A pretreatment. These results suggest that granzyme C rapidly induces target cell death by attacking nuclear and mitochondrial targets and that these targets are distinct from those used by granzyme B to cause classical apoptosis.

Tumor Suppressor NM23-H1 Is a Granzyme A-Activated DNase during CTL-Mediated Apoptosis, and the Nucleosome Assembly Protein SET Is Its Inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.

Dynamic mechanism of nick recognition by DNA ligase.

DNA ligases are the enzymes responsible for the repair of single-stranded and double-stranded nicks in dsDNA. DNA ligases are structurally similar, possibly sharing a common molecular mechanism of nick recognition and ligation catalysis. This mechanism remains unclear, in part because the structure of ligase in complex with dsDNA has yet to be solved. DNA ligases share common structural elements with DNA polymerases, which have been cocrystallized with dsDNA. Based on the observed DNA polymerase-dsDNA interactions, we propose a mechanism for recognition of a single-stranded nick by DNA ligase. According to this mechanism, ligase induces a B-to-A DNA helix transition of the enzyme-bound dsDNA motif, which results in DNA contraction, bending and unwinding. For non-nicked dsDNA, this transition is reversible, leading to dissociation of the enzyme. For a nicked dsDNA substrate, the contraction of the enzyme-bound DNA motif (a) triggers an opened-closed conformational change of the enzyme, and (b) forces the motif to accommodate the strained A/B-form hybrid conformation, in which the nicked strand tends to retain a B-type helix, while the non-nicked strand tends to form a shortened A-type helix. We propose that this conformation is the catalytically competent transition state, which leads to the formation of the DNA-AMP intermediate and to the subsequent sealing of the nick.

Induction of a DNA Nickase in the Presence of Its Target Site Stimulates Adaptive Mutation in Escherichia coli

Adaptive mutation to Lac(+) in Escherichia coli strain FC40 depends on recombination functions and is enhanced by the expression of conjugal functions. To test the hypothesis that the conjugal function that is important for adaptive mutation is the production of a single-strand nick at the conjugal origin, we supplied an exogenous nicking enzyme, the gene II protein (gIIp) of bacteriophage f1, and placed its target sequence near the lac allele. When both gIIp and its target site were present, adaptive mutation was stimulated three- to fourfold. Like normal adaptive mutations, gIIp-induced mutations were recA(+) and ruvC(+) dependent and were mainly single-base deletions in runs of iterated bases. In addition, gIIp with its target site could substitute for conjugal functions in adaptive mutation. These results support the hypothesis that nicking at the conjugal origin initiates the recombination that produces adaptive mutations in this strain of E. coli, and they suggest that nicking may be the only conjugal function required for adaptive mutation.