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At least 19 recordsLinked to original sources
PubMed1976

Structure of a nicked DNA-protein complex isolated from simian virus 40: covalent attachment of the protein to DNA and nick specificity.

A portion of the nicked circular DNA isolated from purified simian virus 40 contains a protein-DNA complex in which protein(s) is covalently attached to the end of a DNA single strand. (Nicked DNA is double-stranded DNA that contains at least one single-strand scission.) The protein was visualized by electron microscopy and labeled in vitro with 125I. The bond between the protein and the DNA is stable in alkali, 4 M guanidine-hydrochloride, 3.86 M hydroxylamine (pH 4,23), and in 98% formamide. Most of the molecules in the nicked circular DNA fraction contained one nick. The nick occurs on either of the two complementary strands; the specific nick sites on the two strands are staggered, but lie within a few hundred nucleotides of each other.

DNA, Circular↗
PubMed1979

Nick translation of mammalian DNA.

The labelling of mouse DNA by nick translation with DNA polymerase I has been investigated with respect to the time of incubation, requirement for DNAase I, size of the product, and uniformity of labelling, and the hybridisability and stability of the resultant labelled probes. Total mouse DNA and reannealed unique mouse DNA sequences can be labelled by nick translation in the presence of [3H]dCTP and [3H]TTP to a specific activity of 7 . 10(6)--20 . 10(6) cpm/microgram DNA. The hybridisation characteristics of nick-translated whole DNA with an excess of unlabelled mouse-embryo driver DNA indicates that no preferential labelling of repetitive or unique DNA sequence classes occurs. In addition, the proportion of unique DNA sequences labelled by nick translation which hybridises with polyadenylated nuclear RNA from Friend cells is the same as that of unique DNA sequences isolated from cells labelled with [3H]thymidine in vivo, indicating that few (if any) of the unique DNA sequences are unrepresented in the nick-translated probe. Probes which contain [3H]dTMP are unstable, and show a considerable reduction in hybridisability over a period of 6 months at --20 degrees C. The decrease is accompanied by an increase in the number of mismatched sites in duplexes containing the labelled probe (as shown by thermal stability measurements of hybrid molecules) and a decrease in the rate of hybridisation of the probe with total mouse DNA. In contrast, DNA which is labelled with [3H]dCMP alone is stable, and does not show any decrease in hybridisability on prolonged storage.

Animals↗
PubMed2025

Methods for purification and characterization of nicked tRNAs.

While tRNA-derived fragments (tDRs) play important roles in gene expression regulation, it is technically challenging to distinguish bona fide tDRs from nicked tRNAs. This is because analytical techniques used to study RNA, such as northern blot, RT-qPCR or sequencing involve the use of denaturing reagents (e.g., phenol, formamide, urea) or physical procedures (e.g., heat) that convert nicked tRNAs into tRNA halves or other tDRs. In this chapter, we describe a protocol that enables the purification of nicked tRNAs under non-denaturing conditions that preserve their 3D structure. Purified nicked tRNAs can then be either enzymatically repaired into almost full-length tRNAs, or chromatographically separated from single-stranded tDRs before detection. These protocols will allow researchers to distinguish between structurally distinct but sequence identical tDRs and nicked tRNAs, disentangling their biological functions.

RNA, Transfer↗
PubMed1979

"Nick translation" in Escherichia coli rep strains deficient in DNA polymerase I activities.

Using phiX1974 replicative form (RF) DNA as an in vivo probe, we have investigated the coordinated action of the 5' leads to 3' exonuclease and polymerase activities of DNA polymerase I in order to understand better its physiological role. We constructed double mutants containing the rep mutation (the replication of phiX174 RF does not occur in rep mutants) together with a mutation affecting DNA polymerase I, either polA12 or polA546ex. Using these mutants, which are believed to be thermosensitive in the polymerase function or the 5' leads to 3' exonuclease function respectively, we studied the kinetics of nick translation at the permissive and non-permissive temperatures in vivo. The substrate was the phiX174 replicative form DNA nicked by the phiX174 gene A protein. E. coli rep polA546ex gave the lowest rate of nick translation, although the ability to perform nick translation, at least as measured by our assay, was still present. E. coli rep polA12 showed a similar low rate at the non-permissive temperature but a rate close to the wild-type level at the permissive temperature. Formation of the parental replicative form molecule in either strain was affected little, even at the restrictive temperature. Our results suggest that DNA polymerase I may not play a major role in ongoing DNA replication.

Coliphages↗
PubMed1979

Nicking-closing activity associated with bacteriophage lambda int gene product.

Integrative recombination of bacteriophage lambda requires the action of the protein Int, the product of the phage int gene. In this paper we show that highly purified Int relaxes supercoiled DNA. The association of this nicking-closing activity with Int is shown by: (i) the cosedimentation of nicking-closing and recombination activities of purified Int, (ii) the parallel inactivation of the two activities in purified Int by both heat and a specific antiserum, and (iii) the alteration of both activities in crude extracts of a strain expressing a mutant int gene. The nicking-closing activity of Int functions in the absence of divalent cations and in the absence of an apparent source of chemical energy. The activity displays no obvious sequence specificity and is inhibited by Mg2+, spermidine, and single-stranded DNA. Int relaxes positive as well as negative supercoils. We present a model for the mechanism of strand exchange that describes how the nicking-closing activity of Int might be used during recombination.

Bacteriophage lambda↗
PubMed1977

Activation of botulinum toxins in the absence of nicking.

The derivative toxins purified from cultures of proteolytic strains of Clostridium botulinum types A and F were found to have been only partially nicked but were fully activated. Trypsinization of C. botulinum type B derivative toxin at pH 6.0 resulted in simultaneous activation and nicking, whereas at pH 4.5, activation preceded nicking. The toxin was split by trypsin at pH 6.0 into two fragments with molecular weights of 112, ooo and 57,000. The toxin contained at least three trypsin-sensitive peptide bonds, one of which was more sensitive than the others at pH 6.0. These results indicate that activation of botulinum toxins by trypsin or endogenous protease (s) is not a direct result of nicking.

Botulinum Toxins↗
PubMed1979

The DNA-protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer.

The broad jost range plasmid, RK2, has been isolated as a DNA-protein relaxation complex. Nicking of the plasmid DNA in the relaxation complex occurs at a single specific site (rlx) located approximately 20 kb away from the origin of DNA replication. A cis-acting function required for plasmid transfer, the presumptive origin of transfer, maps in the same region as rlx. The region of RK2 encompassing rlx has been cloned onto pBR322 and shown to promote mobilization of the hybrid plasmid by an RK2 derivative. These results indicate that the RK2 relaxation complex nicks at or near the origin of transfer of the RK2 plasmid.

Bacterial Proteins↗
PubMed1977

Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme.

A target protein for nalidixic and oxolinic acids in Escherichia coli, the nalA gene product (Pnal), was purified to homogeneity as judged by gel electrophoresis, using an in vitro complementation assay. It is a dimer of identical 110,000-dalton subunits. A polypeptide of this molecular weight is uniquely induced by a lambda nalA transducing phage, thereby showing that the purified Pnal is a product of the nalA gene. Nalidixic and oxolinic acids inhibit DNA gyrase activity and induce formation of a relaxation complex analogue. Treatment of the complex with sodium dodecyl sulfate causes a doublestrand break in the DNA substrate and the resulting linear molecule seems covalently bound to protein. Complex formation, unlike the introduction of supertwists, does not require ATP or relaxed circular DNA and is insensitive to novobiocin. DNA gyrase from a strain with a nalA mutation conferring drug resistance (nalA(r)) is 1/100 as sensitive to oxolinic and nalidixic acids with respect to inhibition of supertwisting and induction of the pre-linearization complex. Addition of Pnal restores drug sensitivity and stimulates DNA gyrase activity. DNA gyrase preparations and Pnal catalyze a third reaction sensitive to nalidixic and oxolinic acids, the ATP-independent relaxation of supertwister DNA. Relaxation by gyrase from nalA(r) cells is drug resistant. The nicking-closing activity is distinct from E. coli omega protein in several properties, including the ability to relax positively supertwisted DNA. We postulate that the nalA gene product occurs in two molecular forms, as Pnal and as a gyrase component. Both forms catalyze nicking-closing, and inhibition of this activity by nalidixic and oxolinic acids may account for the inhibition of DNA synthesis by these drugs.

Bacterial Proteins↗
PubMed1979

Nicking-closing enzyme assembles nucleosome-like structures in vitro.

The four core histones (H2A, H2B, H3, and H4) and DNA were assembled into nucleosome-like particles at physiological ionic strengths either by an extract of chromatin rich in nicking-closing activity or by the purified nicking-closing enzyme itself. When histone-DNA complexes were assembled in vitro from relaxed circular DNA, nearly physiological numbers of superhelical turns were induced in the DNA molecule. Electron microscopy of the complexes assembled by the chromatin extract revealed a beaded structure and a reduction of the contour length compared to free DNA. Micrococcal nuclease digestion of the histone-DNA complexes yielded 145-base-pair DNA fragments typical of nucleosome core particles and shorter subnucleosomal DNA fragments of discrete length.

Animals↗
PubMed1978

Purification of a DNA nicking-closing enzyme from mouse L cells.

A DNA nicking-closing enzyme has been purified from the nuclei of mouse L cells to 90% homogeneity. The denatured and reduced form of the enzyme has a molecular weight of 68,000 which is in agreement with the molecular weight of the native enzyme as determined by gel filtration and by sucrose sedimentation velocity assuming the protein is globular. Therefore, the active form of the enzyme is a monopolypeptide. Its isoelectric point is pH 4.2 +/- 0.2. The nicking-closing activity does not require a cofactor and does not involve any sulfhydryl group. The enzyme requires 0.2 M NaCl and pH in the range of 6.5-7.5 for optimal activity.

Cell Nucleus↗
PubMed1977

Single-strand nicking of DNA in vitro by neocarzinostatin and its possible relationship to the mechanism of drug action.

Neocarzinostatin, a protein antibiotic with anti-tumor activity was found to place single-strand scissions in DNA in an in vitro reaction. The drug's cutting activity was strongly dependent on the presence of 2-mercaptoethanol or dithiothreitol but some cutting did take place in the absence of reducing agent at very high drug levels and prolonged incubation. The requirement for reducing agents could not be replaced with NAD+, FAD, NADH or H2O2 and the strand-scission reaction was not affected by Mg2+, EDTA or intercalating agents. Similar profiles of heat-inactivation of neocarzinostatin were found whether activity was measured by the scission of DNA strand either in vitro or in HeLa cells treated with the drug. Furthermore, both of these parameters corresponded closely with the ability of the modified drug to inhibit DNA synthesis and growth of HeLa cells. By column isoelectric focusing it was shown that all four activities are associated with the same protein band (pH 3.28). From these data we conclude that the cytotoxic activity of neocarzinostatin and the nicking of DNA strands in vitro appear to reside in the same protein.

Antibiotics, Antineoplastic↗
PubMed1977

A DNA nicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties.

Vaccinia virus cores contain an activity which is able to relax both left-and right-handed superhelical DNA. This virus-specific nicking closing enzyme has been highly purified and differs from the corresponding host enzyme in salt optimum, in sedimentation coefficient, and in polypeptide composition as determined on sodium dodecyl sulfate/polyacrylamide gels. The enzyme is probably newly synthesized after the cessation of host protein synthesis which follows virus infection. The most highly purified preparation contains two polypeptides, one of molecular weight 24,000 and the other 35,000. The former polypeptide is a major constituent of the virus (7% of total protein by weight), whereas the latter is present in a much smaller amount (0.2%). Chromatography with denatured DNA-cellulose reveals that the activity is predominately associated with those fractions enriched in the polypeptide of greater molecular weight.

DNA, Circular↗
PubMed1979

Nick-free formation of reciprocal heteroduplexes: a simple solution to the topological problem.

Because the individual strands of DNA are intertwined, formation of heteroduplex structures between duplexes--as in presumed recombination intermediates--presents a topological puzzle, known as the winding problem. Previous approaches to this problem have assumed that single-strand breaks are required to permit formation of fully coiled heteroduplexes. This paper describes a simple, nick-free solution to the winding problem that satisfies all topological constraints. Homologous duplexes associated by their minor-groove surfaces can switch strand pairing to form reciprocal heteroduplexes that coil together into a compact, four-stranded helix throughout the region of pairing. Model building shows that this fused heteroduplex structure is plausible, being composed entirely of right-handed primary helices with Watson-Crick base pairing throughout. Its simplicity of formation, structural symmetry, and high degree of specificity are suggestive of a natural mechanism for alignment by base pairing between intact homologous duplexes. Implications for genetic recombination are discussed.

Base Sequence↗
PubMed1979

Nicking-closing enzyme is associated with SV40 DNA in vivo as a sodium dodecyl sulfate-resistant complex.

A fraction of the cellular nicking-closing (NC) enzyme cosediments with SV40 chromatin isolated after Triton X-100 treatment of infected cells nuclei. Extraction of viral DNA according to the Hirt procedure by treatment of infected cells with sodium dodecyl sulfate (SDS) followed by sedimentation in sucrose gradient to separate the DNA from the bulk of detergent also revealed NC activity associated with DNA. Reconstitution experiments showed that only prebinding of the NC enzyme to DNA protects it against irreversible inactivation by SDS. These results suggest that a fraction of the cellular NC activity is indeed associated with the viral chromosome in vivo.

Chromatin↗
PubMed1978

DNA nicking--closing activity from salmon testis.

Salmon testis is a good source of DNA nicking--closing (N--C) enzyme, as expected for rapidly proliferating cells. Partial purification was obtained but it resulted in an unstable form of N--C enzyme. Cruder fractions are useful for relaxing supercoiled DNA under conditions where other N--C enzymes are inactive. The enzyme is very tolerant of salt concentration, with activity still detectable at 0.6 M NaCl. It is also active at low temperatures with reasonable activity at 0 degrees C. However, at temperatures greater than 25 degrees C it is rapidly inactivated. Otherwise, its properties are similar to other eucaryotic N--C enzymes.

Animals↗
PubMed1978

The purification and characterization of a DNA nicking-closing enzyme from Bacillus megaterium.

Although several eucaryote DNA nicking--closing (N--C) enzymes have been characterized, only the Escherichia coli enzyme has been extensively studied amongst procaryotes. The latter enzyme is distinctly different from the eucaryotic enzymes and we have therefore purified the N--C enzyme from Bacillus megaterium to determine if procaryotes form a distinctive class of N--C enzymes. The purified B. megaterium N--C enzyme has a molecular weight of 120,000, only partly relaxes negative supercoils, does not affect positive supercoils, requires Mg2+, and is inhibited by 0.2 M KCl. The enzyme is also inhibited by 1 mM nalidixic or oxolinic acids but unaffected by novobiocin. A crude N--C enzyme preparation from Micrococcus luteus shows very similar properties.

Bacillus megaterium↗
PubMed2025

Analysis of metal-dependent DNA nicking activities by Cas endonucleases.

CRISPR-Cas systems use RNA-guided CRISPR-associated (Cas) effectors to neutralize infections in bacteria and archaea. In class 2 CRISPR-Cas systems, Cas9 and Cas12 are single-protein Cas effectors that target double-stranded DNA based on complementarity to the guide RNA before cleaving the target DNA using metal-dependent endonuclease domains. Cas9 and Cas12 proteins can be readily programmed to target any DNA of interest by changing the guiding RNA sequence and have been co-opted for genome editing and other biotechnology purposes. The effect of metal ion concentration is an essential consideration in the physiological role of Cas immunity effectors as well as the biotechnological applications of Cas endonucleases. In this chapter, we describe methods for studying the effect of variable divalent metal ion conditions on the DNA binding and cleavage activities of well-studied Cas9 and Cas12a proteins.

CRISPR-Cas Systems↗