Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared. Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA.     

Detection of carcinogen-induced DNA breaks by nick translation in permeable cells

A nick-translation reaction with $\text{E. coli}$ DNA polymerase I (pol. I) was used to detect $\text{in situ}$ DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [³H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens.     

Filamentous phage replication initiator protein gpII forms a covalent complex with the 5' end of the nick it introduced

Rolling circle type DNA replication is initiated by introduction of a nick in the leading strand of the origin by the initiator protein, which in most cases binds covalently to the 5' end of the nick. In filamentous phage, however, such a covalent complex has not been detected. Using a suitable substrate and short reaction time, we show that filamentous phage initiator gpII forms a covalent complex with nicked DNA, which rapidly dissociates unless gpII is inactivated. A peptide-DNA complex was isolated from trypsin digest of the complex by ion-exchange column chromatography and gel filtration, and its peptide sequence was determined. The result indicated that gpII was linked to DNA by the tyrosine residue at position 197 from the N-terminus. The mutant protein in which this tyrosine was replaced by phenylalanine did not show any detectable activity to complement gene II amber mutant phage in vivo. In vitro, the mutant protein recognized the origin and bent DNA as well as the wild-type does, but failed to introduce a nick and to relax the superhelicity of cognate DNA.     

Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization

A rapid method for localizing large numbers of complete cosmids by nonradioactive in situ hybridization is described. The cosmids are nick translated in the presence of biotin-16-dUTP, incubated with an excess of sonicated human DNA, and used as a probe for in situ hybridization. Sites of hybridization are detected by successive treatments with FITC-labeled avidin and biotinylated anti-avidin antibody. Fifty-two cosmids were localized on chromosome 16 in 5 d relative to translocation breakpoints contained in two cell lines. Rapid identification of chromosome 16 was achieved by cohybridization with a chromosome 16-specific centromeric repeat probe.     

Two nicking enzyme systems specific for mismatch-containing DNA in nuclear extracts from human cells.

We have identified two novel enzyme systems in human HeLa nuclear extracts that can nick at specific sites of DNA molecules with base mismatches, in addition to the T/G mismatch-specific nicking enzyme system (Wiebauer, K., and Jiricny, J. (1989) Nature 339, 234-236). One enzyme (called all-type) can nick all eight base mismatches with different efficiencies. The other (A/G-specific) nicks only DNA containing an A/G mismatch. The all-type enzyme can be separated from the T/G-specific and A/G-specific nicking enzymes by Bio-Rex 70 chromatography. Further purification on a DEAE-5PW column separated the A/G-specific nicking enzyme from the T/G-specific nicking enzyme. Therefore, at least three different enzyme systems are able to cleave mismatched DNA in HeLa nuclear extracts. The all-type and A/G-specific enzymes work at different optimal salt concentrations and cleave at different sites within the mismatched DNA. The all-type enzyme can only cleave at the first phosphodiester bond 5' to the mispaired bases. This enzyme shows nick disparity to only one DNA strand and may be involved in genetic recombination. The A/G-specific enzyme simultaneously makes incisions at the first phosphodiester bond both 5' and 3' to the mispaired adenine but not the guanine base. This enzyme may be involved in an A/G mismatch-specific repair similar to the Escherichia coli mutY (or micA)-dependent pathway.     

Chloroplast RNA polymerase from spinach: purification and DNA-binding proteins

Spinach DNA dependent RNA polymerase was purified from isolated chloroplasts by two different procedures. Analysis of the protein composition of the two preparations by SDS-polyacrylamide gel electrophoresis always shows six abundant polypeptides with Mr of 150, 110, 102, 80, 75 and 38 Kd and one less abundant polypeptide of 25 Kd. Some other proteins ranging from 40–70 Kd in Mr are also detected but in a minor and variable amount. The two preparations have an optimum of enzyme activity at 30°C and at 15 mM (NH₄)₂SO₄ when tested with denatured calf thymus DNA. Binding experiments with two different nick translated fragments of spinach chloroplast DNA show that the 80 and 75 Kd polypeptides possess a strong DNA binding capacity.     

Characterization of an endonuclease activity associated with Friend-murine leukemia virus

An endonuclease activity shown to be associated with Friend leukemia virus has been characterized using double-stranded phi X174 DNA as substrate. In the presence of Mg2+, the endonuclease activity was able to convert supercoiled circular DNA duplexes to the relaxed form by introducing single-stranded nicks into the DNA. Most of the nicked DNA duplexes contained only one nick per strand, since unit length DNA was the predominant species obtained when the nicked DNA was analyzed by alkaline sucrose gradient centrifugation. The regions into which the nick could be introduced were evenly distributed around the circular DNA molecule. When Mn2+ was substituted for Mg2+ in the reaction mixture, the number of nicks introduced into circular DNA duplexes by the virus associated endonuclease was greatly increased. In contrast to circular duplexes, linear duplexes and single-stranded DNA functioned poorly as substrates for the virus-associated enzyme. The Friend leukemia virus-associated endonuclease activity is with respect to these characteristics very similar to the endonuclease activity associated with the p32 protein of the avian myeloblastosis virus [1]. The molecular weight of the Friend leukemia virus endonuclease was estimated by gel filtration on a Sephacryl S-200 column to be about 45 000.     

DMBA induced DNA damage and repair in mammary epithelial cells in vitro measured by a nick translation assay

A new E. coli DNA polymerase I directed nick translation assay was used for measuring 7,12-dimethylbenz[a]anthracene-induced in situ DNA damage and repair in mouse mammary epithelial cells in monolayer culture. The nick translation assay was capable of detecting a DMBA-dose dependent significant increase of DNA damage, and the same assay also allowed monitoring of the DNA repair activity provoked by DMBA treatment of the epithelial cells. This relatively simple method thus provides a rapid assay for carcinogen-induced in situ DNA damage and repair in an epithelial cell tumorigenic system.     

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.     

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G·T mismatch was achieved by placing a nick 10–40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.     

Regulation of bacteriophage f1 DNA replication. I. New functions for genes II and X

Gene II protein is required for all phases of filamentous phage DNA synthesis other than the conversion of the infecting single strand to the parental double-stranded molecule. It introduces a specific nick into the double-stranded replicative form DNA, is required for the initiation of (+) strand synthesis and is responsible for termination and ring closure of the (+) strand product. Here we show that the gene II protein also promotes minus strand synthesis later in infection. Over-expression of gene II protein can induce the conversion of all nascent single-stranded phage DNA to the double-stranded form, even in the presence of the single-stranded DNA-binding gene V protein that would normally sequester the newly synthesized single strands. We also present evidence that the gene X protein (separately translated from an initiator codon within gene II, and identical to the C-terminal one-third of the gene II protein) is a powerful inhibitor of phage-specific DNA synthesis in vivo.     

Two DNA-binding and nick recognition modules in human DNA ligase III

Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bind specifically to nicked DNA over intact duplex DNA. One of these modules comprises the zinc finger domain and DNA-binding domain, which function together as a single DNA binding unit. The catalytic core of ligase III is the second DNA nick-binding module. Both binding modules are required for ligation of blunt ended DNA substrates. Although the zinc finger increases the catalytic efficiency of nick ligation, it appears to occupy the same binding site as the DNA ligase III catalytic core. We present a jackknife model for ligase III that posits conformational changes during nick sensing and ligation to extend the versatility of the enzyme.     

Nick translation of HeLa cell nuclei as a probe for locating DNase I-sensitive nucleosomes

The technique of nick translation of nuclei (Levitt, A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) has been used in HeLa cells to label DNase I-sensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion.     

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.     

Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels.

Poly A containing RNA isolated from embryonic chick calvaria was transferred from 6% formaldehyde 0.75% agarose gels to diazobenzyloxymethyl paper and the paper then hybridized to either nick translated pro alpha 1 collagen cDNA clones, pCg1 or pCg54, or to the nick translated pro alpha 2 collagen cDNA clone, pCg45. From the mobilities of the bands hybridizing most strongly to each, pro alpha 2 collagen mRNA was shown to be slightly larger than pro alpha 1 mRNA; they are 5100 and 4900 nucleotides long respectively. pCg54 also hybridized weakly to two bands of lower mobility, corresponding to RNAs 6.4 and 5.6 kb long. Neither pCg54 nor pCg45 hybridized to type II procollagen mRNA in poly A containing RNA isolated from embryonic chick sterna.     

Structural basis for nick recognition by a minimal pluripotent DNA ligase

Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-A crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO4 nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a beta-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO4 termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.     

Bypass of a nick by the replisome of bacteriophage T7

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.     

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)     

Purification and characterization of UV endonucleases I and II from murine plasmacytoma cells

Three endonucleases from murine plasmacytoma cells that specifically nick DNA which was heavily irradiated with ultraviolet (UV) light were resolved by Sephacryl S-200 column chromatography. Two of these, UV endonucleases I and II, were purified extensively. UV endonuclease I appears to be a monomeric protein with a molecular mass of 43 kDa; UV endonuclease II has an S value of 2.9 S, with a corresponding molecular mass estimated at 28 kDa. Both enzymes act as a class I AP endonuclease, cleaving phosphodiester bonds via a beta-elimination mechanism, so as to form an unsaturated deoxyribose at the 3' terminus. Both have thymine glycol DNA glycosylase activity and their substrate specificities generally appear to be overlapping but not identical. UV endonuclease I acts on both supercoiled and relaxed DNAs, whereas II acts only on supercoiled DNA. Both enzymes are active in EDTA, but have different optima for salt, pH, and Triton X-100. Each enzyme is also present in cultured diploid human fibroblasts.