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PubMed2000

Oxidative damage and induced mutations in m13mp2 phage DNA exposed to N-nitrosopyrrolidine with UVA radiation.

N:-Nitrosopyrrolidine (NPYR) is carcinogenic in rodents and undergoes alpha-hydroxylation upon microsomal CYP450 metabolism, giving rise to mutations. Previously, we reported the direct mutagenicity of NPYR, under ultraviolet A (UVA) irradiation, towards Salmonella typhimurium and phage M13mp2. In the present study, we measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in a replicative form of M13mp2 DNA exposed to NPYR plus UVA. Formation of 5-hydroxy-2'-deoxycytidine in calf thymus DNA treated with NPYR plus UVA was also observed. Singlet oxygen is likely to account for the formation of 8-oxodGuo. We analyzed the spectrum of mutations in lacZalpha of M13mp2 phages produced on transfecting Escherichia coli with the replicative form of phage DNA that had been treated with NPYR plus UVA. The role of oxidative DNA damage in mutagenesis was explored using mutM-proficient and -deficient E.coli strains as the hosts. A higher level of mutation was observed with the mutM-deficient host than with the -proficient host. Base substitutions at GC pairs predominated in both mutM-proficient and -deficient hosts. With the mutM-deficient host, we observed an overall increase in the percentage of GC-->TA transversions. In addition we noted that there were fewer GC-->AT transitions than in the mutM-proficient host. With these hosts, different hot spots were observed and a new GC-->TA hot spot was produced. The formation of 8-oxodGuo in DNA, which is known to induce GC-->TA transversion, may contribute to mutagenesis by NPYR plus UVA.

Animals↗

PubMed1982

The Escherichia coli dnaC gene product. III. Properties of the dnaB-dnaC protein complex.

The Escherichia coli dnaB and dnaC proteins form a tight complex in the presence of ATP (Wickner, S., and Hurwitz, J., (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925). The complexed dnaC protein is resistant to inhibition by the sulfhydryl reagent, N-ethylmaleimide. This protection is not observed when ATP is substituted by AMP, ADP, adenyl 5'-yl imidodiphosphate, or adenosine-5'-O-(3-thiotriphosphate); dATP provides partial protection. A sedimentation coefficient of 15.2 S determined by glycerol gradient sedimentation and a Stokes radius of 64 A determined by gel filtration suggests a molecular weight in the range of 400,000. The complex isolated by DEAE-cellulose chromatography contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons per dnaB protein hexamer (300,000 daltons) consistent with a calculated weight of 474,000. The isolated dnaB-dnaC protein complex functions in vitro in the replication of phage phi X174 single-stranded DNA to the duplex replicative form. Tritium-labeled dnaC protein, absent from an isolated prepriming com-dnaC protein, absent from an isolated prepriming complex intermediate, was nevertheless bound to the phiX replicative form DNA synthesized in vitro. These results suggest that stable inclusion od dnaC protein in the priming complex bound to DNA requires a completely assembled primosome.

Bacterial Proteins↗

PubMed1978

Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and phiX174 DNA: alignment with restriction enzyme maps.

Escherichia coli RNA polymerase has been shown to bind to a limited number of Hind and Hae III restriction enzyme fragments. On S13 replicative form DNA there are three major binding sites, and the locations correlate with promoter sites at the beginning of genes a and B and a site overlapping gene D and the beginning of gene E. Two less definite binding sites have been localized, one in gene F and one at the gene G-H junction. In phiX174 replicative form DNA, five sites, each with apparently similar binding properties, have been located, four of which correspond exactly to binding sites in S13. One site, at the beginning of the B gene, could not be assigned to exactly the same location found in S13. This was due in part to differences in the restriction cleavage maps in the area of the DNA and possibly to the higher background of nonspecific binding in the phiX174 experiments. The location of two of the phiX174 sites at the beginning of genes A and D-E corresponds very well with transcription data, but the site at the start of the B gene indicates the promoter site is closer to the initiation sequence of the B protein than was previously suggested on the basis of transcription data.

Binding Sites↗

PubMed1981

Restriction map of the single-stranded DNA genome of Kilham rat virus strains 171, a nondefective parvovirus.

We constructed a physical map of Kilham rat virus strains 171 DNA by analyzing the sizes and locations of restriction endonuclease-generated fragments of the replicative-form viral DNA synthesized in vitro. BglI, KpnI, BamHI, SmaI, XhoI, and XorII did not appear to have any cleavage sites, whereas 11 other enzymes cleaved the genome at one to eight sites, and AluI generated more than 12 distinct fragments. The 30 restriction sites that were mapped were distributed randomly in the viral genome. A comparison of the restriction fragments of in vivo- and in vitro-replicated replicative-form DNAs showed that these DNAs were identical except in the size or configuration of the terminal fragments.

Chromosome Mapping↗

PubMed1980

Replication of duplex DNA of phage phi X174 reconstituted with purified enzymes.

Replication of the covalently closed duplex replicative form (RF) of phage phi X174 DNA has been achieved by coupling two known enzyme systems: (i) synthesis of viral strand circles (SS) from RF, and (ii) conversion of SS to nearly complete RF (RF II). In this coupled system, activated RF (gene A . RF II complex) was a more efficient template and generated as many as 10 RF II molecules per RF input, at a rate commensurate with SS synthesis. The 11 proteins required for the two component systems were all needed in the coupled RF duplication system; no new factors were required. Single-stranded DNA binding protein was needed for RF duplication at only 4% the level needed in its stoichiometric participation in SS synthesis. In addition to RF II, more complex replicative forms appeared late in the reaction, and their possible origin is discussed.

Bacteriophage phi X 174↗

PubMed1986

Synthesis of bacteriophage phi X174 in vitro: mechanism of switch from DNA replication to DNA packaging.

Replication of a replicative form DNA of bacteriophage phi X174 initiates by rolling-circle synthesis of the viral DNA followed by discontinuous synthesis of the complementary DNA. Gene C protein of phi X174, which is involved in DNA packaging, inhibits the rolling-circle DNA synthesis by binding to the initiation complex in vitro. The gene C protein-associated initiation complex can synthesize and package the viral DNA to produce infectious phage when supplemented with phi X174 gene J protein and the prohead. Multiple rounds of phage synthesis occur without dissociation of the gene C protein from the complex. These results indicate that gene C protein is central in the switch from replication of a replicative form DNA to synthesis and concomitant packaging of viral DNA into phage capsid, which occurs in the late stage of infection.

Bacteriophage phi X 174↗

PubMed2000

Human immunodeficiency virus type 1 quasi species that rebound after discontinuation of highly active antiretroviral therapy are similar to the viral quasi species present before initiation of therapy.

In an effort to identify the sources of the viruses that emerge after discontinuation of therapy, analyses of human immunodeficiency virus (HIV) quasi species were done for 3 patients with sustained levels of HIV RNA of <50 copies/mL for 1-3 years. The sequences found in the rebounding plasma virus were closely related to those of the actively replicating form of viruses present before the initiation of combination therapy. All quasi species found in the rebounding plasma virus were also present in proviral DNA, cell-associated RNA in peripheral blood mononuclear cells (PBMC), and virion RNA derived from PBMC coculture during periods when plasma HIV RNA levels were <50 copies/mL. These findings suggest that the rapid resurgence of plasma viremia observed after discontinuation of therapy and the viruses cocultured from PBMC are derived from a relatively stable pool of the replicating form of virus rather than from activation of a previously latent pool.

Adult↗

PubMed1982

Adenovirus DNA replication in vivo: properties of short DNA molecules extracted from infected cells.

Adenovirus type 5 (Ad5) DNA replicating in intact HeLa cells was pulse-labeled with [3H]thymidine extracted by the procedure of Hirt and analyzed on neutral sucrose gradients. In addition to viral replicative forms (over 33 kb), slowly sedimenting species of DNA (0.05-3 kb) was observed. Hybridization analysis showed that this DNA contained about 30% Ad5 DNA sequences. Analysis of the sensitivity of this DNA to the 5' OH-specific spleen exonuclease after alkali or RNAase treatment revealed that about 80% of these molecules contained ribonucleotides. DNA in this fraction was labeled at the 5' end with 32P and hybridized together with control 3H-labeled Ad5 DNA to Hpa I restriction fragments immobilized on nitrocellulose paper. Many of these small molecules were located near the termini. Alkali treatment prior to hybridization decreased the 32P/3H ratio throughout the genome. This suggests that some of these Ad5 molecules possess ribonucleotides and therefore may be intermediates in discontinuous DNA replication. Longer molecules (over 0.5 kb) were found sedimenting with viral replicative forms and mature DNA. The Ad5 molecules in this fraction showed no evidence of alkali-labile termini.

Adenoviridae↗

PubMed1995

Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity.

The hepatitis B virus (HBV) envelope contains equimolar amounts of three viral proteins: the major (S), middle, and large (L) polypeptides. Their roles in the adsorption and penetration of the virus have not yet been elucidated. We have used a highly efficient in vitro model that permits reproducible HBV infection to investigate whether N-myristylation, a posttranslational modification of the L protein, was essential for viral infectivity. A point mutation abolishing myristylation was introduced into the HBV genome. Mutant virions were produced by transfecting viral DNA into hepatoma cells and their infectivity was evaluated on primary human hepatocyte cultures. No difference between mutant and wild-type viral RNA production could be observed. Furthermore, intermediate DNA replicative forms were observed in transfected cells demonstrating replication competence of mutant viral genomes. In addition, complete virions were produced in the cell supernatant. However, we found that mutant viral particles contained viral DNA with a reduced mean size, probably corresponding to a larger single-stranded region in the relaxed circular DNA form. We have evidenced the presence of pre-S1, pre-S2, and S epitopes at the outer surface of these virions by using immunoprecipitation with specific monoclonal antibodies. This result confirmed that mutant viruses were normally assembled. By contrast, myristylation-defective mutants completely lost their infectivity for human hepatocytes in primary cultures as shown by the absence of HBs antigen production and viral intermediate replicative forms in hepatocytes. In conclusion, the myristylation of the L protein is not required for the production of Dane-like particles but it is absolutely necessary for HBV infectivity.

Alanine↗

PubMed1990

Hepatitis B virus DNA patterns in the liver of children with chronic hepatitis B.

The pattern of hepatitis B virus DNA (HBV-DNA) expression were studied in 2 sequential liver biopsies from 26 children (18 treated with interferon and 8 controls) with chronic hepatitis B. In the basal biopsy replicative forms of HBV-DNA were detected in all of the samples and integrated viral DNA was present in 1 case. At the end of the study, 8 children had lost serum HBV-DNA although 2 of the children were still HBeAg positive. (Six had been treated with interferon.) In all of the cases, HBV-DNA was not detectable in the final biopsy. For the rest of the patients, HBV-DNA was positive in serum and all of them had replicative forms of HBV-DNA in the second liver sample. None of the patients lost hepatitis B surface antigen (HBsAg). Peripheral blood mononuclear cells (PBMC) from these patients were studied. HBV-DNA was not found in the PBMC of the 8 children without serum HBV-DNA, and HBV-DNA was detected in the PBMC of 5/12 patients with serum HBV-DNA. In conclusion, HBV-DNA disappeared from the biopsies of children who lost circulating HBV-DNA, although some of the patients were still HBeAg positive. This result implies that the detection of HBV-DNA in liver is important in order to assess the efficacy of the antiviral therapy. On the other hand, HBsAG remained positive in all children at the end of the study although HBV-DNA was not detected in serum, liver, and PBMC by the conventional hybridization techniques.

Adolescent↗

PubMed1975

Initiation of transcription within an RNA-polymerase binding site.

1. The 5'-terminal sequence of the RNA transcribed from bacteriophage fd replicative form DNA under the control of promotor region I has been determined to be ppp(Gp)nUpApApApGpApCpCpUpGpApUpUp. . . 2. This sequence is complementary to the 5'-terminal sequence of the minus strand of the corresponding RNA polymerase binding site I, the starting point for RNA synthesis lying approximately in the middle of the binding site. 3. This initial sequence is also transcribed faithfully from isolated complexes of RNA polymerase and binding site I, obtained by DNase digestion of complexes between RNA polymerase and fd replicative form DNA. These highly stable complexes can not be reconstituted from binding site and enzyme. 4. It is concluded that RNA polymerase binding site and initiation site are identical parts of a promoter region, and that no "drift" between these sites is required as a step in RNA chain initiation. An additional non-transcribed outside region is implicated as essential for full promoter function.

Base Sequence↗

PubMed1989

Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles.

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.

Animals↗

PubMed2001

Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules.

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA-DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.

Bacteriophage M13↗

PubMed1970

Process of infection with bacteriophage phiX174. XXXV. Cistron 8.

Twenty-two new amber and ochre mutants of phiX174 were isolated and classified into complementation groups. Three ochre mutants gave positive complementation tests with reference mutants in the seven previously defined groups and thus represent an eighth cistron. Studies of the physiology of infection in the nonpermissive condition for mutants in cistron VIII yielded the following information. (i) Replicative-form synthesis proceeds at a normal rate, and is turned off at the usual time. (ii) Synthesis of single-stranded deoxyribonucleic acid (DNA) is delayed until nearly 40 min after infection (in the absence of lysis), at which time a slow synthesis of infectious phage particles commences. The synthesis of infectious particles at late times is interpreted as a consequence of "leakage," and indicates that the cistron VIII product is required in very small quantities. (iii) During the normal period of single-strand synthesis, most of the replicative-form DNA is found in a form with properties similar to those of the transient intermediates of single-strand DNA synthesized during normal infection.

Carbon Isotopes↗

PubMed1989

Parvovirus replication in normal and transformed human cells correlates with the nuclear translocation of the early protein NS1.

The parvovirus H-1 infection of the normal human diploid fibroblast strain MRC-5 produces a cytopathic effect, but no increase in infectious virus has been observed. Previously, we reported that large amounts of empty capsids are assembled in the nucleus of H-1 infected MRC-5 cells (S. Singer and S. Rhode, in D. Ward and P. Tattersall, ed., Replication of Mammalian Parvoviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1978). The level of viral replicative-form DNA synthesis as shown by metabolic labeling is markedly reduced in these cells. Synthesis of the early protein NS1 is normal or slightly decreased, and the usual amount of the 92,000-molecular-weight (92K) posttranslationally modified NS1 was seen. The second deficient parameter that we have observed in the abortive infection is the nuclear translocation of NS1. In contrast, the simian virus 40-transformed MRC-5 cell line MRC-5 V1 and the simian virus 40-transformed human kidney cell line NB undergo a productive infection by H-1 accompanied by more efficient translocation of NS1 to the nucleus. The results indicate that there is an association between defective translocation of the NS1 rep protein to the nucleus and defective amplification of parvovirus replicative-form DNA. The nuclear translocation of specific proteins seems to be a function that is altered by development or neoplastic transformation.

Capsid↗

PubMed1997

The small envelope protein is required for secretion of a naturally occurring hepatitis B virus mutant with pre-S1 deleted.

Naturally occurring deletions in the hepatitis B virus pre-S1 domain have been frequently found during persistent viral infection. In this study we have investigated the functional properties of a mutant viral genome that carries an in-frame deletion of 183 nucleotides in the pre-S1 region. This deletion removes the promoter of the small envelope gene. Transfection into human hepatocellular carcinoma cells of a replication-competent construct containing this deletion resulted in an increase of intermediate DNA replicative forms compared to those produced by wild-type hepatitis B virus. Northern blot analysis revealed that such cells lack the 2.1-kb transcripts encoding the small envelope protein and that hepatitis B surface antigen was absent as well. Furthermore, nucleocapsids containing the genome with pre-S1 deleted were not secreted, and the deleted large envelope protein was retained with the cytoplasm and exhibited a perinuclear pattern of distribution. However, coexpression with the small envelope protein was sufficient to restore virion secretion and to change the cellular distribution of the deleted large envelope protein. In addition, the creation of point mutations that prevent the synthesis of large or small envelope proteins also inhibited viral secretion and led to increased levels of hepatitis B virus intermediate replicative forms within the cell. These studies suggest that naturally occurring viral mutants with pre-S1 deletions involving the promoter region of the small envelope gene will generate a deleted large envelope protein that is retained in the endoplasmic reticulum, resulting in the accumulation of nucleocapsids containing viral DNA; transcomplementation with the wild-type small envelope protein will allow mutant virion secretion to occur.

Animals↗

PubMed1979

The use of Haemophilus gallinarum DNA-relaxing enzyme to investigate the relationship between the number of superhelical turns and the molecular weight in a negatively twisted DNA.

Covalently closed-circular, superhelical DNAs, including viral DNAs, bacterial plasmid DNAs, and bacteriophage replicative-form DNA, were treated with a small amount of Haemophilus gallinarum DNA-relaxing enzyme to generate incompletely relaxed DNA molecules. Each sample consisted of a set of closed-circular DNA molecules differing by one turn in their number of superhelical turns. The DNA samples were analyzed by agarose gel electrophoresis under conditions such that the electrophoretic mobility was a function of the number of turns. The numbers of superhelical turns (at 37 degrees C in 20 mM Tris-HCl (pH 7.5)-5 mM MgCl2) in the DNAs of pSC101 (5.8 megadaltons), Colicin E1 (4.2 megadaltons), pMR4 (4.0 megadaltons; recombinant between pBR322 and lambda DNA fragment), phi X174 replicative-form (RF) I, Simian virus 40 (SV40), and polyoma virus (3.4--3.6 megadaltons each), and lambda dv021 (2.05 megadaltons) were estimated to be 36, 27, 23--24, 20--21, 20--21, 20--21, and 11--13, respectively. It appears that the number of superhelical turns is mainly a function of the molecular weight of the DNA, at least in the substrates tested here.

Chromatography, Agarose↗

PubMed1996

Inactivation by phthalocyanine photosensitization of multiple forms of human immunodeficiency virus in red cell concentrates.

BACKGROUND: The use of phthalocyanines in conjunction with red light has been shown to inactivate model lipid-enveloped viruses in red cell concentrates. The ability of this treatment to inactivate multiple forms of human immunodeficiency virus (HIV) was evaluated in this study. STUDY DESIGN AND METHODS: The phthalocyanines used were aluminum phthalocyanine tetrasulfonate (AIPcS4) and the silicon phthalocyanines HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), and HOSiPcOSi(CH3)2 (CH2)3N+(CH3)3I-(Pc 5). HIV was studied in a cell-free form, in an actively replicating form, in latently infected cells, and in blood from HIV-positive patients. RESULTS: All three phthalocyanines inactivate > or = 10(5) infectious doses of cell-free HIV. However, only Pc 4 effectively inactivated actively replicating HIV and latently infected cells. The latter was about four times as sensitive to inactivation as was actively replicating HIV. Increasing the hematocrit of red cells during treatment decreased the rate of inactivation, especially at lower light doses. Under treatment conditions that completely inactivated the laboratory isolates of HIV, cell-associated HIV in blood from HIV-positive patients was also completely inactivated. The polymerase chain reaction signal from the gag gene of HIV was not affected on treatment of cell-free virus, but it was reduced after treatment of cell-associated HIV, particularly in some latently infected cell lines. CONCLUSION: Pc 4 and red light are effective in eliminating the infectivity of HIV in red cell concentrates. The usefulness of this approach for blood banking depends on future demonstration of the preservation of red cell circulatory survival and function in vivo.

Base Sequence↗