共查询到20条相似文献,搜索用时 0 毫秒
1.
2.
3.
Studies on the biological role of DNA methylation: inhibition of methylation and maturation of the bacteriophage phichi174 by nicotinamide. 总被引:4,自引:4,他引:4
下载免费PDF全文

Nicotinamide was found to be a potent inhibitor of DNA methylation in vivo without interfering with protein or DNA synthesis. The inhibition of DNA methylation in a phage-infected cell resulted in a parallel decrease in the production of viable virus particles. In vitro experiments revealed that nicotinamide inhibits DNA methylase activity in a competitive fashion with respect to S-adenosylmethionine and non-competitively with respect to DNA. These results were interpreted to mean that DNA methylation is an essential step in the process of maturation of the bacteriophage phichi174. 相似文献
4.
5.
Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins. Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination). In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin. When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins. Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination. In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products. This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave. Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined. With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence. Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred. 相似文献
6.
7.
A progressive degradation of the parental viral strand label is observed upon infection of a Rep- mutant of Escherichia coli by 32P-labeled phiX174. Very little parental label remains in the RF (replicative form) by 47 min after infection. Concomitant with this degradation, replicative intermediates are formed which sediment at 21s, the rate of RF I (supercoiled-closed circular DNA), in a neutral sucrose gradient but which denature and sediment in alkaline gradients as single strands of unit size and larger. These denaturable 21s replicative intermediates have been shown previously to be RF molecules containing an elongated viral strand. Addition of chloramphenicol at 7 min after infection at 30 mug/ml, a concentration sufficient to block RF leads to SS (single strand) synthesis but not RF leads to RF synthesis in a wild-type host cell, reduced the amount of viral strand elongation but did not prevent viral strand degradation. The addition of chloramphenicol at 150 mug/ml at 7 min after infection totally prevents both the degradation of the parental label and the formation of the replicative intermediates with elongated tails. We infer that degradation of the viral strand requires the gene A-mediated nicking of the viral strand but not the concomitant elongation of the viral strand. 相似文献
8.
9.
W T Hsu E J Lin P P Fu R G Harvey S B Weiss 《Biochemical and biophysical research communications》1979,88(1):251-257
The ØX174 DNA assay system developed earlier is utilized to determine the comparative reactivities with nucleic acid of the diolepoxide metabolites of a series of polycyclic aromatic hydrocarbons varying in carcinogenic potency. The infectious ØX174 viral DNA is exposed to the hydrocarbon derivative for 10 min., then infectivity of the treated DNA is assayed by incubation with spheroplasts, counting plaque formation on agar plates. The bay region diolepoxides of benzo[a]-pyrene, chrysene, and dibenz[a,h]anthracene, implicated as the ultimately active carcinogenic metabolites of the parent hydrocarbons, exhibit potent viral inhibitory activity. On the other hand no correlation is evident between viral inhibitory activity and either the location of the diolepoxide function in a bay region or the theoretically calculated β-delocalization energies (ΔEdeloc.) of the carbonium ion arising from opening the epoxide ring. The significance of these findings with respect to theories of carcinogenesis is discussed. 相似文献
10.
Studies on the biological role of DNA methylation: V. The pattern of E.coli DNA methylation. 总被引:10,自引:2,他引:10
下载免费PDF全文

The distribution of the methylatable sites GATC and CCATGG was studied by analyzing the molecular average size of restriction fragments of E. coli DNA. Both sites were found to be randomly distributed, reflecting a random pattern of methylation. The methylation pattern of specific sequences such as the origin of replication and rRNA genes has been studied in wild type E. coli and a methylation deficient (dam- dcm-) mutant. These sequences were found to be methylated in wild type cells and unmethylated in the mutant indicating that there is no effect of the state of methylation of these sequences on their expression. Analysis of the state of methylation of GATC sites in newly replicating DNA using the restriction enzyme Dpn I (cleaves only when both strands are methylated) revealed no detectable hemimethylated DNA suggesting that methylation occurs at the replication fork. Taking together the results presented here and previously published data (5), we arrive at the conclusion that the most likely function of E. coli DNA methylations is probably in preventing nuclease activity. 相似文献
11.
12.
13.
14.
Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA
+
recA
+) and HF4704 (uvrA
-
recA
+), but not in the recombination deficient strain HF4712 (uvrA
+
recA
-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA
+ but persisted within the recA
- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA
+
recA
+, but slightly reduced in uvrA
+
recA
- and severely suppressed in uvrA
-
recA
+ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome. 相似文献
15.
Studies on the biological role of dna methylation; IV. Mode of methylation of DNA in E. coli cells 总被引:11,自引:9,他引:11
下载免费PDF全文

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues. 相似文献
16.
T Dokland R A Bernal A Burch S Pletnev B A Fane M G Rossmann 《Journal of molecular biology》1999,288(4):595-608
An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching. 相似文献
17.
18.
Enzymatic synthesis of duplex circular phiX174 DNA containing phosphoramidate bonds in the (-) strand.
下载免费PDF全文

Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution. 相似文献
19.
Rifampin inhibition of bacteriophage phiX174 parental replicative-form DNA synthesis in an Escherichia coli dnaC mutant. 总被引:2,自引:5,他引:2
下载免费PDF全文

The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage phiX174 parental replicative-form synthesis (Kranias and Dumas, 1974). However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, phiX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in phiX174 parental replicative-form DNA synthesis, presumably in its initiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of phiX174 DNA are discussed. 相似文献