The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks.

The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .     

Arabinosylcytosine-induced accumulation of DNA nicks in myotube nuclei detected by in situ nick translation

This laboratory has recently reported the occurrence of DNA nicking at the onset of terminal skeletal myogenesis by using the technique of in situ nick translation (Dawson and Lough: Dev. Biol., 127:362-367, 1988). Because 1-beta-D-arabinofuranosylcytosine (araC), a cytocidal agent that is routinely used to removed dividing fibroblasts from myogenic cultures, inhibits DNA repair, it was of interest to determine whether araC treatment resulted in an accumulation of the endogenously created nicks. Thus, we have assessed the accumulation of DNA nicks in myotube cells during a 20 hour araC treatment period at the onset of terminal myogenesis (44-64 hours in vitro) by using three techniques: alkaline sucrose gradient density centrifugation, kinetic in situ nick translation, and cellular in situ nick translation. Although alkaline sucrose gradient centrifugation revealed no detectable nicking after 20 hours, kinetic in situ nick translation analysis revealed subtle but significant increases in DNA nicks caused by araC within 7 hours of drug application, and a 1.5-fold increase in DNA repair sites after 20 hours of drug treatment. That these observations reflected nicking specifically in myotube nuclei was determined by immunocytochemical localization of nicked sites after repair with a biotinylated nucleotide analog (biotin-11-dUTP). The effects of araC were only incompletely reversible, whether or not the drug was removed from the cultures, within 2 days of the treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation

Summary A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.     

Mapping of eukaryotic DNA topoisomerase I catalyzed cleavage without concomitant religation in the vicinity of DNA structural anomalies

Sensitive sites for covalent trapping of eukaryotic topoisomerase I at DNA structural anomalies were mapped by a new method using purified enzyme and defined DNA substrates. To insure that the obtained topoisomerase I trapping patterns were not influenced by DNA sequence variations, a single DNA imperfection was placed centrally within a homonucleotide track. Mapping of topoisomerase I-mediated irreversible cleavage sites on homopolymeric DNA substrates containing mismatches showed trapping of the enzyme in several positions in close vicinity of the DNA imperfection, with a strong preference for the 5' junction between the duplex DNA and the base-pairing anomaly. On homopolymeric DNA substrates containing a nick, sites of topoisomerase I-mediated cleavage on the intact strand were located just opposite to the nick and from one to ten nucleotides 5' to the nick. Sites of enzyme-mediated cleavage next to a nick and an immobile single-stranded branch were located 5' to the strand interruption in distances of two to six nucleotides and two to ten nucleotides, respectively. Taken together these findings suggest that covalent trapping of topoisomerase I proceeds at positions adjacent to mismatches, nicks and single-stranded branches, where the cleavage reaction is allowed and the ensuing ligation reaction prevented. In principle, the developed interference method might be of general utility to define topoisomerase-DNA interactions relative to different types of structural anomalies.     

Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein.

RepC protein encoded by plasmid pT181 has single-stranded endonuclease and topoisomerase-like activities. These activities may be involved in the initiation (and termination) of pT181 replication by a rolling circle mechanism. RepC protein cleaves the bottom strand of DNA within the origin of replication at a single, specific site when the DNA is in the supercoiled or linear (double or single-stranded) form. We have found that RepC protein will also cleave single-stranded DNA at sites other than the origin of replication. We have mapped the secondary cleavage sites on pT181 DNA. When the DNA is in the supercoiled, or linear, double-stranded form, only the primary site within the origin is cleaved. However, when the DNA is present in the single-stranded form, several strong and weak cleavage sites are observed. The DNA sequence at these cleavage sites shows a strong similarity with the primary cleavage site. The presence of Escherichia coli SSB protein inhibited cleavage at all of the secondary nick sites while the primary nick site remained susceptible to cleavage.     

Photoaffinity labelling of atrial natriuretic factor (ANF)-R1 receptor by underivatized 125I-ANF. Involvement of lipid peroxidation.

A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate.     

Mechanism of strand passage by Escherichia coli topoisomerase I. The role of the required nick in catenation and knotting of duplex DNA

We studied the interaction between topoisomerase I and a nicked DNA substrate to determine how the nick permits Escherichia coli topoisomerase I to catenate and knot duplex DNA rings. The presence of just a single nick in a 6600-base pair DNA increased the amount of DNA bound to topoisomerase I by 6-fold. The enzyme acts at the nick, as shown by linearization of nicked circles and covalent attachment of an enzyme molecule opposite the nick. DNA breaks are also introduced by the enzyme at sites not opposite to a nick, but three orders of magnitude less efficiently. The break induced by the enzyme is within several base pairs of the nick and on the complementary strand, but the exact site cut is dictated by DNA sequence requirements. Because these sequence requirements are identical to those for cutting of single-stranded DNA, we conclude that the enzyme stabilizes a denatured region at the nick. Breaks in single-stranded DNA occur 98% of the time when a C residue is four bases to the 5' side unless G is adjacent and 5' to the break. For a DNA circle nicked at a unique location, the efficiency of DNA breakage opposite the nick correlates with the rate of catenation. We present a unified model for the relaxation, catenation, and knotting reactions of topoisomerase I in which the enzyme induces a break in a single-stranded region, but bridges that break with covalent and noncovalent interactions and allows passage of one duplex or single-stranded DNA segment.     

Expression,purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase

We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg²⁺ and Mn²⁺. The optimal concentrations of ATP cofactor and Mg²⁺ ion were 0.01–2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5′ to the nick inhibited ligation more than those at the first position 3′ to the nick. The mismatches at other positions 5′ to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5′ to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5′ to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.     

Human mismatch repair and G*T mismatch binding by hMutSalpha in vitro is inhibited by adriamycin, actinomycin D, and nogalamycin

Loss of the human DNA mismatch repair pathway confers cross-resistance to structurally unrelated anticancer drugs. Examples include cisplatin, doxorubicin (adriamycin), and specific alkylating agents. We focused on defining the molecular events that link adriamycin to mismatch repair-dependent drug resistance because adriamycin, unlike drugs that covalently modify DNA, can interact reversibly with DNA. We found that adriamycin, nogalamycin, and actinomycin D comprise a class of drugs that reversibly inhibits human mismatch repair in vitro at low micromolar concentrations. The substrate DNA was not covalently modified by adriamycin treatment in a way that prevents repair, and the inhibition was independent of the number of intercalation sites separating the mismatch and the DNA nick used to direct repair, from 10 to 808 base pairs. Over the broad concentration range tested, there was no evidence for recognition of intercalated adriamycin by MutSalpha as if it were an insertion mismatch. Inhibition apparently results from the ability of the intercalated drug to prevent mismatch binding, shown using a defined mobility shift assay, which occurs at drug concentrations that inhibit repair. These data suggest that adriamycin interacts with the mismatch repair pathway through a mechanism distinct from the manner by which covalent DNA lesions are processed.     

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.     

Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.     

Genotoxicity of formaldehyde and an evaluation of its effects on the DNA repair process in human diploid fibroblasts

Formaldehyde treatment of human fibroblasts gave rise to DNA damage detected by a nick translation assay. This damage was not repaired by typical 'long-patch'-type excision repair as evidenced by the failure of DNA repair inhibitor post-treatment to elevate the amount of DNA strand breakage. In addition, the effects of formaldehyde on DNA repair were examined in light of a recent report suggesting that formaldehyde inhibited the repair of X-ray-induced strand breaks and UV- and benzo [a]pyrene diol epoxide-induced unscheduled DNA synthesis in human bronchial cells. We report that formaldehyde (1) was ineffective at inhibiting the sealing of X-ray- or bleomycin-induced DNA strand breaks, (2) did not inhibit the removal of pyrimidine dimers from cellular DNA at short treatment times, and (3) that the previously observed inhibition of unscheduled DNA synthesis was most likely due to the inhibition of uptake of labeled precursor into formaldehyde-treated cells. Thus, our findings are not consistent with the notion that formaldehyde inhibits the repair process in human fibroblasts. Finally, formaldehyde was shown to elevate the level of misincorporation of bases into synthetic polynucleotides catalyzed by E. coli DNA polymerase I, indicating that the mutagenicity of formaldehyde may be due to covalent alteration of DNA bases.     

A novel biotinylated adenylate analogue derived from pyrazolo[3,4-d]pyrimidine for labeling DNA probes.

A novel dATP analogue, 3-[5-[(N-biotinyl-6- amiocaproyl)amino]pentyl]-1-(2-deoxy-beta-D-erythro-pentofuranosyl )-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5'-triphosphate (9, bio-13-dAPPTP), which is modified at the 3-position with a flexible linker arm bearing a terminal biotin moiety, has been synthesized. This nucleotide is readily incorporated into DNA probes by nick translation. These probes hybridize to complementary targets as well as probes labeled with bio-dUTP, as judged by slot blot. When incorporated into oligonucleotides, they do not cause the loss of hybridization efficiency that an N-6-substituted adenine nucleotide does when incorporated into the same sites in the oligonucleotide.     

Mechanisms of in situ nick translation of chromosomes using restriction endonucleases

In situ nick translation of mammalian chromosomes by restriction endonuclease treatment to nick the chromosomal DNA, and 'translation' in the presence of DNA polymerase I and biotinylated dUTP, results in a distinct banding pattern. Further experiments have elucidated the mechanisms producing these bands. The hypothesis is presented that differences in the local conformation of the DNA-protein complex, rather than the DNA sequence itself, lead to the nick translation bands. The different DNase I sensitivity along the chromosomes suggests that the bands, which were clearly evident, reflect morphological units closely related to biological functions.     

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.     

Error‐free and mutagenic processing of topoisomerase 1‐provoked damage at genomic ribonucleotides

Genomic ribonucleotides incorporated during DNA replication are commonly repaired by RNase H2‐dependent ribonucleotide excision repair (RER). When RNase H2 is compromised, such as in Aicardi‐Goutières patients, genomic ribonucleotides either persist or are processed by DNA topoisomerase 1 (Top1) by either error‐free or mutagenic repair. Here, we present a biochemical analysis of these pathways. Top1 cleavage at genomic ribonucleotides can produce ribonucleoside‐2′,3′‐cyclic phosphate‐terminated nicks. Remarkably, this nick is rapidly reverted by Top1, thereby providing another opportunity for repair by RER. However, the 2′,3′‐cyclic phosphate‐terminated nick is also processed by Top1 incision, generally 2 nucleotides upstream of the nick, which produces a covalent Top1–DNA complex with a 2‐nucleotide gap. We show that these covalent complexes can be processed by proteolysis, followed by removal of the phospho‐peptide by Tdp1 and the 3′‐phosphate by Tpp1 to mediate error‐free repair. However, when the 2‐nucleotide gap is associated with a dinucleotide repeat sequence, sequence slippage re‐alignment followed by Top1‐mediated religation can occur which results in 2‐nucleotide deletion. The efficiency of deletion formation shows strong sequence‐context dependence.     

Phosphatidylcholine and phosphatidylethanolamine enhance the activity of the mammalian mitochondrial endonuclease in vitro

The purified endonuclease of bovine heart mitochondria extensively degrades a variety of DNA templates in vitro but shows a remarkably strong preference to nick within one specific evolutionarily conserved sequence block of 12 consecutive guanine residues which resides just upstream from the heavy strand origin of mitochondrial DNA replication (Low, R. L., Cummings, O. W., and King, T. C. (1987) J. Biol. Chem. 262, 16164-16170). If the enzyme serves to provide an important nicking function at this site in vivo, then mitochondrial factors likely exist which further enhance the enzyme's recognition of this locus and prevent cleavage at other less favored sites. In this study, we report that specific membrane phospholipids appear to exert such effects in vitro. In standard endonuclease assays, low levels of phosphatidylcholine or phosphatidylethanolamine (0.5 mM) stimulate the purified enzyme activity 10-20-fold. However, at moderate levels (20-40 mM), these phospholipids largely inhibit widespread degradation of duplex DNA while still allowing site-specific nicking at the conserved guanine target in the mitochondrial genome. These findings suggest that an interaction of the endonuclease with major lipid components of the inner membrane could be an important determinant of the enzyme's specificity for mitochondrial DNA.     

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.