Finding of a zero linking number topoisomer

It is generally assumed that native deoxyribonucleic acid (DNA) is a right-handed double helix. A reasonable deduction is that during replication, the two parental strands have to unwind very quickly. However, this surmised quick unwinding is problematic and has never been proven experimentally. It is hypothesized that the two strands of DNA are winding with each other ambidextrously rather than plectonemically. The successful assembling and disassembling of a zero linking number topoisomer supports this hypothesis. It was further proven by quick separation of singly nicked DNA. The new DNA model was also verified by the “figure 8” structure, which is the annealing product of two single-stranded circular DNA with a 2 kb complementary insert in opposite directions. These experimental results are hard to be explained by the traditional Watson–Crick model. The significance of this finding in the understanding of DNA replication is briefly discussed.     

Mechanism of mitochondrial DNA replication in mouse L-cells: asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence

Examination of in vivo long-labeled, pulse-labeled and pulse-chase-labeled mitochondrial DNA has corroborated and extended the basic elements of the displacement model of replication. Mitochondrial DNA molecules are shown to replicate an average of once per cell doubling in exponentially growing cultures. Analysis of the separate strands of partially replicated molecules indicates that replication is highly asynchronous with heavy-strand synthesis preceding light-strand synthesis. Native and denatured pulse-labeled replicating molecules exhibit sedimentation properties predicted by the displacement model of replication. Pulse-label incorporated into molecules isolated in the lower band region of ethidium bromide/cesium chloride gradients is found primarily in heavy daughter strands. Pulse-label incorporated into molecules isolated in the upper band region is found primarily in light daughter strands. The results of a series of pulse-chase experiments indicate that the complete process of replication requires approximately 120 minutes. Both daughter molecules are shown to segregate in an open circular form. They are then converted to closed circular molecules having a superhelix density near zero. After closure, the 7 S heavy-strand initation sequence is synthesized, and this process is accompanied by nicking, unwinding and closing of at least one of the parental strands resulting in the formation of the D-loop structure. The 7 S heavy-strand initiation sequence of the D-loop structure is not stable and turns over with a half-life of 7·9 hours. We suggest that all in vivo forms of parental closed circular mitochondrial DNA have superhelix densities of near zero, and that the previously observed superhelix density of closed circular mitochondrial DNA, σ~ ?0·02, results from the loss of the 7 S heavy-strand initiation sequence from D-loop mitochondrial DNA molecules during isolation.     

Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences

The relative immunogenicities of the poly[d(G-C)] and poly[d(A-C) · d(G-T)] families of helices have been determined. The specificities of the resultant immunoglobulins have been characterized for recognition of different synthetic and natural left-handed sequences and conformations. Certain modifications of poly[d(G-C)] in the sugar-phosphate bacbone and cytosine C-5 potentiate the right(R)-to-left(L) (B → Z) transition under physiological conditions. The resulting polynucleotides, poly[d(G^S-C)], poly[d(G-io⁵C)], poly[d(G-br⁵C)] and poly[d(G-m⁵C)], are also highly immunogenic. In contrast, DNAs incapable of assuming the left-handed conformation under physiological salt concentrations are weakly or non-immunogenic. These include unmodified poly[d(G-C)] as well as members of the poly[d(A-C) · d(G-T)] family of sequences bearing pyrimidine C-5 substitutions (methyl, bromo, iodo). These polynucleotides undergo the R → L isomerization under more stringent ionic and thermal conditions.The specificities of purified polyclonal and monoclonal anti-Z DNA immunoglobulins (IgG) were measured by binding to radiolabeled polynucleotides, by electrophoretic analysis of IgG bound to covalent closed circular DNAs, and by immunofluorescent staining of polytene chromosomes. The salt-induced left-handed forms of poly[d(G-C)] and its derivatives (including the cytidine C-5 methyl, bromo, iodo, and N-5 aza substituted polynucleotides) and of the modified poly[d(A-C) · d(G-T)] polymers are bound to varying degrees by different antibodies. The patterns of substrate recognition demonstrate the existence of several antigenic domains in left-handed DNAs, including the helix convex surface and the sugar-phosphate backbone. Substitutions in these regions can produce enhancing (required substitutions), neutral, or inhibitory effects on subsequent IgG binding. Additionally, certain modifications of either the convex surface of Z DNA at the C-5 position of cytidine (i.e. a methyl group) or of the backbone (i.e. phosphorothioate substitution) can lead to polymorphic lefthanded conformations that are compatible with antibody binding when present individually but not in combination. The recognition patterns exhibited with DNA substrates from the two DNA families indicate that some, but not all, IgGs show specificity for different nucleotide sequences.The anti-Z DNA IgGs were used to probe for specific left-handed Z DNA determinants on plasmid (e.g. pBR322) or viral (e.g. simian virus 40 (SV40)) DNAs and on the acid-fixed polytene chromosomes of dipteran larvae. At their extracted superhelical density, the negatively supercoiled form I, but not the relaxed, nicked, or linear forms of all tested plasmid and viral DNAs specifically bind sequence-independent anti-Z IgGs. Dimers, trimers and higher oligomers of form I DNA cross-linked by bivalent anti-Z IgGs are formed with numerous (e.g. φX174, SV40, pBR322) genomes. Their occurrence depends upon IgG concentration and specificity, the conditions of ionic strength and temperatures and the DNA genome. The IgG cross-linked DNA multimers are converted to monomers by dithiothreitol reduction. Sequence-independent monovalent anti-Z Fab fragments bind form I DNA but do not generate oligomeric species. Multimers of order >2 indicate the existence of at least two anti-Z Ig binding sites per molecule, as in the case of SV40. IgGs differ in their ability to form stable complexes with some sites on natural DNAs, presumably due to their sequence and conformation binding specificities. A differential binding of these antibodies is also observed in certain bands of polytene chromosomes, such as the telomeric regions that are involved in chromosome associations.     

5-bromodeoxyuridine labeling of monomeric and catenated circular mitochondrial DNA in HeLa cells

Bromouracil labeling of the mitochondrial DNA in exponentially growing HeLa cells produces two hybrid mitochondrial DNA species, with density shifts of 41.9 and 54.0 mg/ml relative to unlabeled mitochondrial DNA, as well as heavy mitochondrial DNA, with a shift of 95.3 mg/ml. The two hybrid species result from the difference in thymine composition of the complementary strands of mitochondrial DNA. In addition, mitochondrial DNA with a density intermediate between the hybrid and unlabeled species was found. This quarter heavy mitochondrial DNA represents 25% (w/w) of the total DNA after eight hours of labeling, and forms two peaks with shifts of 20.6 and 27.0 mg/ml relative to unlabeled mitochondrial DNA. 70% (w/w) of the quarter heavy mitochondrial DNA is in catenated forms, while 30% (w/w) is monomeric. Degradation of the catenanes by shearing of purified quarter heavy mitochondrial DNA results in the appearance of hybrid and unlabeled mitochondrial DNA bands, demonstrating that the quarter heavy catenanes contain both hybrid and unlabeled submolecules. The implications of the structure of the quarter heavy catenanes on the mechanism of formation of catenanes are discussed.     

Complex structure of the membrane nuclease of Streptococcus pneumoniae revealed by two-dimensional electrophoresis

Close contacts between Escherichia coli RNA polymerase and specific purine residues in the tryptophan (trp) operon promoter of Salmonella typhimurium were revealed using the methylating agent dimethyl sulfate. RNA polymerase bound to trp promoter DNA caused alterations in the rate of methylation at seven specific sites; in the anti-sense strand, guanine residues at positions ?37, ?34 and ?2 showed enhanced methylation, while those at positions ?14, ?6 and +3 showed reduced methylation. In the sense strand, only the guanine residue at ?32 showed reduced methylation. No RNA polymerase contacts with adenine residues were observed. Using the same method, close interactions between E. coli trp repressor and purine residues in the trp operator of S. typhimurium were examined. Bound trp repressor alters the methylation rates of both guanine and adenine residues from positions ?25 to +3. The points of contact are distributed rather symmetrically on both DNA strands. Three points of close contact are shared by RNA polymerase and trp repressor, supporting previous models of trp repressor action.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Biogenesis of mitochondria: XXV. Studies on the mitochondrial genomes of petite mutants of yeast using ethidium bromide as a probe

This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ^?ER^r) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ^?ER^r clones, and in both the gene elimination rate is slower than in the parent ρ⁺ ER^r strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes.