DNA methylation in wheat. Purification and properties of DNA methyltransferase

The origin and function of the large amount of 5-methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300-fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE-cellulose, Sephadex G-75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in double-stranded DNAs isolated from wheat, maize, calf thymus or bacteria using S-adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de novo and in maintenance methylation. A relative molecular mass of 50,000-55,000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of Mr = 50,000 and one other component (Mr = 35,000). The preference for endogenous, double-stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA.     

Exogenous DNA transcription in cells with their native DNA inhibited. 1. DNA incorporation into homologous and heterologous cells.

The incorporation of mouse S-EAC DNA into homologous normal cells (mouse embryo secondary cultures), and into heterologous cancer cells (TC-SV40 line), with both systems having their native DNA blocked by BrUdR incorporation, was studied. 3H-TdR-DNA was inoculated with DEAE-D to protect it and to potentiate its incorporation, the process being autoradiograohically controlled. The amount of incorporated DNA was radioisotopically determined, and the incorporation process was studied by analysing the fractions obtained after density gradient centrifugation separation of the inoculated cells DNA. Receptivity was greater in those cells inoculated with DEAE-D-protected DNA. The incorporation was slightly greater for cells whose DNA had been blocked by BrUdR incorporation, and for homologous with respect to heterologous cells. In those cells inoculated while the DNA blockade was incomplete, part of the inoculated DNA became incorporated into the cell genome (L-H chains). However, in the completely blocked cells it could not be determined if the incorporation occurred in a lysogenic-like or in an episomic-like form.     

DNA topoisomerase I cleavage sites in DNA and in nucleoprotein complexes.

The intracellular substrate for eukaryotic DNA topoisomerases is chromatin rather than protein-free DNA. Yet, little is known about the action of topoisomerases on chromatin-associated DNA. We have analyzed to what extent the organization of DNA in chromatin influences the accessibility of DNA molecules for topoisomerase I cleavage in vitro. Using potassium dodecyl sulfate precipitation (Trask et al., 1984), we found that DNA in chromatin is cleaved by the enzyme with somewhat reduced efficiency compared to protein-free DNA. Furthermore, using native SV40 chromatin and mononucleosomes assembled in vitro, we show that DNA bound to histone octamer complexes is cleaved by topoisomerase I and that the cleavage sites as well as their overall distribution are identical in histone-bound and in protein-free DNA molecules.     

DNA methylation and the frequency of CpG in animal DNA.

An analysis of nearest neighbour dinucleotide frequencies and the level of DNA methylation in animals strongly supports the suggestion that 5-methylcytosine (5mC) tends to mutate abnormally frequently to T. This tendency is the likely cause of the CpG deficiency in heavily methylated genomes.     

DNA in profile.

The double helix structure of DNA is not necessarily straight but rather can be curved at almost every base pair. Thus, each piece of DNA possesses a unique silhouette, as individual as its base sequence.     

Unmethylated DNA in mouse L cells. I. DNA fibre autoradiography.

Enzymatic methylation of DNA in mouse L cells has been studied using DNA fibre autoradiography to analyse the distribution of 5-methylcytosine in chromosomal DNA. The autoradiographic pattern of DNA labelled in the 5-methylcytosine is in several respects similar to the pattern of DNA replication. Two mean features are apparent: (1) the silver grains appear in well defined sections, and (2) the labelled sections are arranged in tandem along each DNA double helix. After a short pulse of radioactivity in the rate of growth of labeled sections in the pattern of DNA replication and the enzymatic methylation of DNA are identical. Unlike the replication pattern, DNA labeled during the S phase with L-[Me-3H] methionine is not completely labeled. There are distinct, 8-20 mum intervals in the autoradiographic pattern of this DNA. The length of these intervals may correspond to unmethylated sections of chromosomal DNA of about 23 to 58 kilo base pairs. These unmethylated sections of chromosomal DNA represent about 10% of the total genome.     

DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Interdomain communication in DNA topoisomerase II. DNA binding and enzyme activation

Topoisomerase II catalyzes the ATP-dependent transport of a DNA segment (T-DNA) through a transient double strand break in another DNA segment (G-DNA). A fundamental mechanistic question is how the individual steps in this process are coordinated. We probed communication between the DNA binding sites and the individual enzymatic activities, ATP hydrolysis, and DNA cleavage. We employed short DNA duplexes to control occupancy at the two binding sites of wild-type enzyme and a variant with a G-DNA site mutation. The DNA concentration dependence of ATP hydrolysis and a fluorescence anisotropy assay provided thermodynamic information about DNA binding. The results suggest that G-DNA binds with higher affinity than T-DNA. Enzyme with only G-DNA bound is competent to cleave DNA, indicating that T-DNA is dispensable for DNA cleavage. The ATPase activity of enzyme bound solely to G-DNA is partially stimulated. Full stimulation requires binding of T-DNA. Both DNA binding sites therefore signal to the ATPase domains. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future mechanistic dissection.     

DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases.

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.     

DNA sequencing and helix-coil transition. I. Theory of DNA melting

An explicit analytic formula accurately describing the melting of a natural DNA is derived. For phage ?X-174 and virus SV-40, the nucleotide sequences of which are known, the formula fits experimental data for the differential melting curve almost within the experimental accuracy.     

Ultraviolet-induced DNA excision repair in human B and T lymphocytes. II. Effect of inhibitors and DNA precursors.

Despite their great sensitivity to ultraviolet light purified human B and T lymphocytes are capable of complete repair provided that the ultraviolet dose does not exceed 0.5 Jm-2. Their capacity to repair, as measured by the restoration of DNA supercoiling in preparations of nucleoids, and their survival are significantly increased in the presence of deoxyribonucleosides. Certain agents which inhibit semi-conservative DNA synthesis (hydroxyurea, 1-beta-D-arabinofuranosylcytosine (arafCyt) either stop or delay the repair process in lymphocytes. The effect of hydroxyurea is eventually overcome spontaneously, but changes in the sedimentation behaviour of ultraviolet-irradiated nucleoids caused by arafCyt can only be neutralized by addition of deoxycytidine. The effective inhibition of repair by arafCyt permits the detection of extremely small amounts of ultraviolet damage and also the estimation of when repair is complete.     

DNA binding and antigenic specifications of DNA gyrase.

Complexes of DNA gyrase and minichromosomal DNA containing the origin of replication of Escherichia coli (oriC) can be formed without metabolic energy and visualised by electron microscopy. The A subunit, part of the A2B2-DNA gyrase complex is the binding protein. Various binding sites are scattered around the minichromosomal DNA including oriC. The minimal origin contains the only prominent and reproducible binding site. Binding to this site is suppressed by oxolinic acid and the ATP analogue beta-y-imido ATP. If gyrase isolated from the gram-positive bacterium Bacillus subtilis is used no binding to oriC is seen. This observation is consistent with antigenic differences between the A subunits of the two microorganisms. The binding to oriC might reflect a requirement for DNA gyrase during the initiation of DNA replication.     

Related base sequences in the DNA of simple and complex organisms. I. DNA/DNA duplex formation and the incidence of partially related base sequences in DNA

A comparative study has been made of the reactions of bacteriophage T4, B. subtilis,and mouse DNA with homologous DNA trapped on a membrane filter. The variation of reaction rate with temperature provides information as to the number of unique and partially related base sequences. The thermal stability of the duplexes formed at various temperatures has been used as a criterion of the extent of base pairing. These studies suggest that no closely related base sequences exist within the T4 genome. Similar results were obtained with Bacillus subtilisDNA although the data suggest the possibility of some very distant intragenome homologies. In contrast, mouse DNA contains so many related base sequences that the predominant reaction product is always one containing partially complementary strands. These results suggest that gene duplication has been a major mechanism operative in the increase of DNA content of metazoans through evolution. The relationship between the existence of partial base sequence redundancies and the mechanism of recombination is also discussed. This research was supported by a grant from the National Science Foundation (GB 6099).     

Fluctuations in superhelical DNA.

The effect of superhelicity on the base-pair opening probability and on the probability of occurrence of cruciform states in palindromic regions is theoretically treated. The calculations show that below the superhelix density value of -sigma=0.05 superhelicity does not appreciably affect the characteristics of DNA secondary structure fluctuations. In the range of physiological superhelix densities sigma (-sigma=0.05-0.09) the base-pair opening probability markedly increases. However, within this range of sigma the base-pairs are opened only transiently and permanently open regions are not formed. Permanently opened regions appear at higher negative superhelix densities (-sigma greater than 0.10). At the values of -sigma higher than 0.06 a cruciform structure in the palindromic region centred in position 3965 proves to be the most probable fluctuational disturbance in the 0x174 duplex DNA. Different experimental approaches used for probing the fluctuations in superhelical DNA have been analysed. The results suggest that most direct quantitative information can be derived from data on the nicking of closed DNA by single strand-specific endonucleases. Such data (Wang, 1974) accord with the results of theoretical calculations. Calculations show that, due to base-pair opening, the total free energy of superhelical DNA should depend parabolically on sigma only up to some critical value of sigma=sigmac. If negative superhelicity exceeds this critical value, which under physiological conditions proves to be -sigma=0.085, the free energy should increase linearly with -sigma. The biological role of supercoiling is discussed in the light of obtained results.     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.