DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.     

In vitro methylation of DNA with Hpa II methylase.

The enzyme Hpa II methylase extracted and partially purified from Haemophilus parainfluenza catalyzes the methylation of the tetranucleotide sequence CCGG at the internal cytosine. The enzyme will methylate this sequence if both DNA strands are unmethylated or if only one strand is unmethylated. Conditions have been developed for producing fully methylated DNA from various sources. In vitro methylation of this site protects the DNA against digestion by the restriction enzyme Hpa II as well as the enzyme Sma I which recognizes the hexanucleotide sequence CCCGGG. These properties make this enzyme a valuable tool for analyzing methylation in eukaryotic DNA where the sequence CCGG is highly methylated. The activity of this methylase on such DNA indicates the degree of undermethylation of the CCGG sequence. Several examples show that this technique can be used to detect small changes in the methylation state of eukaryotic DNA.     

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.     

DNA methylase from HeLa cell nuclei.

A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG.     

DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.     

Substrate specificity of Ca2+,Mg2+-dependent DNAase from sea urchin (Strongylocentrotus intermedius) embryos

Ca2+,Mg2+-dependent DNAse from sea urchin embryos is specific to the secondary structure of substrates irrespective of the nature of activating cations. The enzyme does not split synthetic single-stranded oligo and polynucleotides, such as d(pTpTpTpCpC), d(pGpGpTpTpT). d(pApApTpTpC), d(pGpApApTpTpC), d(pA)5-poly(dT), d(pApApTpTpC)-poly(dT), poly(dA) and poly (dT) and hydrolyses the double-stranded substrates poly d(AT), poly (dA) . poly (dT) and highly polymerized DNA. Native double-stranded DNA from salmon and phage T7 is split by the enzyme at a higher rate than that of denaturated DNA of salmon and single-stranded DNA of phage M13. The high rate of poly(dA) . poly(dT) and poly d(AT) hydrolysis and the stability of poly(dG) . poly(dC) to the effect of the enzyme suggest a certain specificity of the enzyme to the nature of nitrogenous bases at the hydrolyzed phosphodiester bond of the substrate.     

Interaction of a cysteine-containing peptide with DNA

Cystine peptide dimer (Lys-Gly-Val-Cys-Val-N2H2Dns)2 with S-S bridge was synthesized and its interactions with DNA and synthetic polynucleotides have been studied by optical spectroscopy methods. By recording fluorescent titration curves we have shown that the affinity of the peptide to different synthetic polynucleotides decreases in the order: poly(dG).poly(dC) greater than poly(dA).poly(dT) greater than poly(dGC).poly(dGC). The stability of complexes to increasing concentrations of NaCl diminishes in the same order. The association constant is about 20-fold greater for peptide binding to poly(dG).poly(dC) than to poly(dA).poly(dT). By using circular dichroism and fluorescence measurements we have shown that the peptide competes for the binding sites on DNA with two minor-groove binding antibiotics--distamycin A and sybiromycin. These results have suggested that the peptide also binds in the DNA minor groove. Investigation of the interactions between such peptides and DNA may be useful for constructing ligands with combined specificity to DNA.     

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.     

Binding of nonintercalative antitumor drugs to DNA-polymers: structural effects of bisquaternary ammonium heterocycles

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA.dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA.dT preference in their binding affinity to DNA. Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC).poly(dG-dC), poly(rA).poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA).poly(dT) and poly(dI).poly(dC) indicating a slow kinetics. The preferred binding to dA.dT base pairs in DNA decreases in the order from SN-61367 greater than SN-13232 greater than SN-6324,SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA.dT).poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAse I cleavage of poly(dA-dT).poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNA binding and dA.dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Binding of CC-1065 to poly- and oligonucleotides

The binding of the antitumor agent CC-1065 to a variety of poly- and oligonucleotides was studied by electronic absorption, CD, and resistance to removal by Sephadex column chromatography. Competitive binding experiments between CC-1065 and netropsin were carried out with calf-thymus DNA, poly(dI-dC) · poly(dI-dC), poly(dI) · poly(dC), poly(rA) · poly(dT), poly(dA- dC) · poly(dG-dT), and poly(dA) · 2poly(dT). CC-1065 binds to polynucleotides by three mechanisms. In the first, CC-1065 binds only weakly, as judged by the induction of zero or very weak CD spectra and low resistance to extraction of drug from the polynucleotide by Sephadex chromatography. In the second and third mechanisms, CC-1065 binds strongly, as judged by the induction of two distinct, intense CD spectra and high resistance to extraction of drug from the polynucleotide, by Sephadex chromatography in both cases. The species bound by the second mechanism converts to that bound by the third mechanism with varying kinetics, which depend both on the base-pair sequence and composition of the polynucleotide. Competitive binding experiments with netropsin show that CC-1065 binds strongly in the minor groove of DNA by the second and third mechanisms of binding. Netropsin can displace CC-1065 that is bound by the second mechanism but not that bound by the third mechanism. CC-1065 binds preferentially to B-form duplex DNA and weakly (by the first binding mechanism) or not at all to RNA, DNA, and RNA–DNA polynucleotides which adopt the A-form conformation or to single-strand DNA. This correlation of strong binding of CC-1065 to B-form duplex DNA is consistent with x-ray data, which suggest an anomalous structure for poly(dI) · poly(rC), as compared with poly(rI) · poly(dC) (A-form) and poly(dI) · poly(dC) (B-form). The binding data indicate that poly(rA) · poly(dU) takes the B-form secondary structure like poly(rA) · poly(dT). Triple-stranded poly(dA) · 2poly(dT) and poly(dA) · 2poly(dU), which are considered to adopt the A-form conformation, bind CC-1065 strongly. Netropsin, which also shows a binding preference for B-form polynucleotides, also binds to poly(dA) · 2poly(dT) and occupies the same binding site as CC-1065. These binding studies are consistent with results of x-ray studies, which suggest that A-form triplex DNA retains some structural features of B-form DNA that are not present in A-form duplex DNA; i.e., the axial rise per nucleotide and the base tilt. Triple-stranded poly(dA) · 2poly(rU) does not bind CC-1065 strongly but has nearly the same conformation as poly(dA) · 2poly(dT) based on x-ray analysis. This suggests that the 2′-OH group of the poly(rU) strands interferes with CC-1065 binding to this polynucleotide. The same type of interference may occur for other RNA and DNA–RNA polynucleotides that bind CC-1065 weakly.     

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.     

Effect of enzymatic methylation on the chemical,physical, and biological properties of DNA

DNA methylase was partially purified from Escherichia coli W and used to methylate DNA from Bacillus subtilis and bacteriophage φ105. The former DNA was methylated 1.17% and the latter 0.87%. The products were 6-methyladenine (85%) and 5-methylcytosine (15%) in both cases. The methylated DNA was stable toward depurination and viscosity loss at elevated temperatures. Methylation led to a 50% decrease in transforming activity in two strains of B. subtilis and no change in a third strain. The ability of phage φ105 DNA to rescue a defective phage strain was decreased 50% by methylation. No changes were observed in the ability of methylated DNA to serve as a template for DNA polymerase or RNA polymerase. The pattern of cleavage of DNA by a variety of restriction endonucleases was not affected by methylation. There were no changes in the physicochemical properties of DNA on methylation as measured by hyperchromicity on heating, formaldehyde denaturation, viscosity, and sedimentation.     

Evidence for template-specific sites in DNA polymerases

Using rabbit hemoglobin messenger RNA as template, $\text{E. coli}$ polymerase I produces poly (dT), poly (dA)·(dT) and antimessenger DNA products. Mild heating of the enzyme causes a differential loss in activity as indicated by three rates of inactivation for the three types of synthesis. Heat inactivation studies have also been carried out with DNA polymerases from oncogenic RNA viruses and mammalian sources using various homopolymer-oligomer pairs as primertemplates. In general, for any given enzyme these synthetic primer-templates reveal different extents of inactivation of the polymerase. These findings may be interpreted to suggest a) that the binding of DNA polymerase to various primer-templates produces conformational changes in the enzyme which are dependent on the type of template bound, or b) that many, if not all, DNA polymerases have different subsites for different templates.