Restriction endonuclease cleavage of linear and closed circular murine leukemia viral DNAs: discovery of a smaller circular form.

Both linear (form III) and closed circular (form I) viral DNAs obtained from mouse cells infected with Moloney murine leukemia virus were cleaved by Sal I, Sma I, Bam HI and Pst I restriction endonucleases. DNA fragments generated by these cleavages were ordered with respect to the 5' and 3' ends of the RNA genome by several techniques, including comparisons of the DNA fragments from cleavages of the linear and closed circular forms, double digestions using different combinations of enzymes and the use of an RNA probe specific for the 3' end. DNA from Hirt extractions of infected cells yielded a discrete species of linear viral DNA whose size was determined by agarose gel electrophoresis to be 5.7 x 10(6) daltons. In the course of characterizing the closed circular DNA, we observed two form I DNA molecules. The larger molecule was the same size as the linear DNA. The second molecule migrated faster on agarose gels and was the predominant species of the two closed circular DNAs. Using the restriction endonuclease maps which we derived, we demonstrate that this novel form I DNA is a smaller homogeneous species of viral DNA, missing about 600 nucleotides found in the linear and larger closed circular DNA molecules. We have localized the site of this missing DNA piece to be at either one or both ends of the linear viral DNA.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

DNA-histone interaction in the vicinity of replication points.

Chromatin replication was studied in isolated nuclei from Concanavalin A activated lymphocytes. Digestion with micrococcal nuclease revealed that the resistant fraction of in vitro replicated DNA is associated with nucleosomes. Earlier experiments had shown that the nuclease resistant fraction of nascent DNA is composed of fragments which are shorter than the nuclease resistant fragments of bulk DNA. In this communication we demonstrate that the short fragments of nascent DNA are differently bound to nucleosome like structures compared to bulk DNA. At 0.5 M NaCl a fraction of pulse labeled labeled DNA is released from these structures and appears as free double stranded DNA of about 140 base pair length (5S DNA) while the 185 pair fragments of mature replicated DNA remain attached to nucleosomes under these conditions. The experiments may indicate that the interaction of a fraction of replicating DNA with histones differs from that of bulk DNA.     

Abnormal synthesis of mitochondrial DNA in the presence of N-methyl-N-nitro-N-nitrosoguanidine in vitro.

1. The in vitro effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on mtDNA synthesis was studied using isolated newborn rat liver mitochondria. 2. From the kinetics of the incorporation of [3H]thymidine into the acid-insoluble material, MNNG neither stimulated nor inhibited the DNA synthesizing activity of mitochondria. The activity observed in the presence of MNNG was inhibited by N-ethylmaleimide and actinomycin D. 3. By the band velocity sedimentation in CsCl/ethidium bromide, the properties of the nascent mtDNA formed in the presence of MNNG were analyzed. The nascent DNA-containing molecule was not found in the closed-circle fraction, and essentially detected in the open-circle fraction. This change of the template was blocked by N-ethylmaleimide but not by actinomycin D, suggesting a conversion of the closed-circular template to the open-circular one by single-strand cleavage(s). From the band sedimentation in alkaline CsCl, the number of nascent higher molecular DNAs was increased but the molecules were all of relatively lower molecular weight. On the other hand, the formation of nascent fragments was inhibited. 4. The alkaline CsCl equilibrium centrifugation analysis revealed that the nascent DNA synthesized in the presence of MNNG consisted of both light and heavy components. 5. Present results suggest that MNNG exerts its effect on the mtDNA synthesis by modifying the intrinsic mechanism of discontinuous synthesis, since the conversion of the template DNA molecule from the closed- to open-circular form and the continuous polymerization of the nascent higher-molecular DNA on such a relaxed template were characteristic events in vivo.     

DNA methylation in mammalian nuclei

A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Identification of novel DNA forms in tomato golden mosaic virus infected tissue. Evidence for a two component viral genome.

Extracts obtained from cells infected with the geminivirus tomato golden mosaic (TGMV) are shown to contain, in addition to viral single-stranded DNA, several novel species of virus-specific single- and double- stranded DNA (ss and ds DNA). The results of nuclease studies and electron microscopy suggest that three of the intracellular DNAs are unit-genome length duplexes of closed circular, relaxed circular, and linear form. The remaining ds DNA species are of high molecular weight and appear to be concatamers consisting of two or more unit-length circular ds TGMV DNA resulted in fragments whose combined size is twice the unit-genome length. Thus ds TGMV is composed of two components of nearly identical size but different nucleotide sequence.     

Molecular cloning of the unintegrated squirrel monkey retrovirus genome: organization and distribution of related sequences in primate DNAs

The closed circular form of the endogenous squirrel monkey type D retrovirus (SMRV) was molecularly cloned in a bacteriophage vector. The restriction map of the biologically active clone was determined and found to be identical to that of the parental SMRV linear DNA except for the deletion of one long terminal repeat. Restriction enzyme analysis and Southern blotting indicated that the SMRV long terminal repeat was approximately 300 base pairs long. The SMRV restriction map was oriented to the viral RNA by using a gene-specific probe from baboon endogenous virus. Restriction enzyme digests of a variety of vertebrate DNAs were analyzed for DNA sequence homology with SMRV by using the cloned SMRV genome as a probe. Consistent with earlier studies, multiple copies of SMRV were detected in squirrel monkey DNA. Related fragments were also detected in the DNAs from other primate species, including humans.     

Joint molecule formation following conjugation in wild type and mutant Escherichia coli recipients

The sedimentation coefficients of closed circular Simian virus (SV40) DNA, phage PM2 DNA and animal mitochondrial DNAs in alkaline NaCl and alkaline CsCl were found to decrease by about 5% as the initial superhelix densities decreased from 0.0 to ?0.10, corresponding to a decrease in the degree of strand interwinding from 1.0 to 0.9 net turns per ten base pairs. The small dependence of the appropriately normalized sedimentation coefficients on the degree of strand interwinding is taken to indicate that fully titrated and denatured closed circular DNA is highly supercoiled in a positive sense. This supercoiling results from the spontaneous decrease in the number of secondary turns in the no longer ordered pairs of polynucleotide strands.The measured sedimentation coefficients form a smoothly connected monotonie curve when plotted along with the sedimentation coefficients in alkali (Sebring et al., 1971) of parental closed circles derived from closed circular SV40 DNA replicating intermediates. These DNAs have degrees of strand interwinding that range from 0.6 to 0.15.The possibility raised by Paoletti &; LePecq (1971) that closed circular duplex DNAs contain positive supercoils, i.e. have degrees of strand interwinding greater than 1.0, has been ruled out in a series of ethidium bromide titrations of partially replicated mitochondrial DNA before and after removal of the progeny strand. More ethidium bromide was required in the latter case for relaxation, a result which shows that intercalated ethidium and a displacing strand act on the duplex in the same way, and that both unwind the duplex. This result requires the supercoils of naturally closed circular DNAs to be negative.     

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.     

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.     

The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity

Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs.The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images     

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.     

Genome analysis of Agrobacterium tumefaciens: construction of physical maps for linear and circular chromosomal DNAs, determination of copy number ratio and mapping of chromosomal virulence genes.

The phytopathogenic bacterium Agrobacterium tumefaciens is unique in that it possesses both linear and circular DNA chromosomes in addition to a plant-tumor-inducing (Ti) plasmid. We analyzed the two chromosomal DNA molecules in strain MAFF301001, whose Ti plasmid has already been sequenced completely. Physical maps of the chromosomal DNAs were constructed by Southern hybridization experiments using Pme I and Swa I fragments and short fragments bridging the Swa I fragments with special care to avoid any missing fragment. Hybridization with 16S rDNA probe showed one rDNA locus on the linear chromosome and two loci on the circular chromosome. For this bacterium to be pathogenic, not only Ti plasmid but also chromosomal genes are required. The chromosomal virulence (chv) genes (chvA, chvB, chvD, chvE, chvG, chvH, and chvI) and the chromosomal genes affecting the virulence [acvB, pgm(exoC), glgP, miaA, and ros] were successfully mapped onto 5 different regions in the chromosomal physical maps. These chv genes and the chromosomal genes affecting the virulence other than pgm and glgP were found on the circular chromosome, whereas the pgm and glgP genes were located on the linear chromosome. In contrast to the large terminal inverted repeats of Streptomyces linear chromosomal DNA, no hybridization signal was detected between left and right terminal fragments of the linear A. tumefaciens chromosome. Quantitative analysis of DNA fragments indicated that the copy numbers of the two chromosomal DNAs and the Ti plasmid are identical.     

Degradation of DNA in cells and extracts of the obligately anaerobic bacterium Roseburia cecicola upon exposure to air

High-molecular-weight chromosomal DNA from Roseburia cecicola, an oxygen-intolerant anaerobe, could be isolated only when the bacterial cells were kept under anaerobic conditions up to the time of cell lysis. When the cells were exposed to oxygen before lysis, the chromosomal DNA degraded. Likewise, linear but not covalently closed circular DNAs degraded in cell extracts of the organism that were exposed to atmospheres containing O2 but not in extracts that were maintained in a reduced state. Covalently closed circular DNAs were nicked but not degraded in the oxidized extracts.     

Degradation of DNA in cells and extracts of the obligately anaerobic bacterium Roseburia cecicola upon exposure to air.

High-molecular-weight chromosomal DNA from Roseburia cecicola, an oxygen-intolerant anaerobe, could be isolated only when the bacterial cells were kept under anaerobic conditions up to the time of cell lysis. When the cells were exposed to oxygen before lysis, the chromosomal DNA degraded. Likewise, linear but not covalently closed circular DNAs degraded in cell extracts of the organism that were exposed to atmospheres containing O2 but not in extracts that were maintained in a reduced state. Covalently closed circular DNAs were nicked but not degraded in the oxidized extracts.     

Pulse-labeled, small closed circular DNA in cultured mouse and human cells

Mouse (erythroleukemia, TSA8, and FM3A) cells and human (HeLa and HL-60) cells were pulse-labeled with [3H]thymidine and covalently closed circular DNA in the extrachromosomal fraction was analyzed by fluorography following polyacrylamide gel electrophoresis. Two discrete bands for mouse and at least one, different, band for human cells emerged in the position to which small circular DNA (less than 1 kb) migrate, suggesting there to be species-specific, preferentially labeled, small circular DNA in mammalian cells. The incorporation of [3H]thymidine into the DNA was inhibited by cycloheximide but unaffected by aphidicolin. Restriction enzyme (AluI) digestion of the DNA fraction from MEL cells produced approximately 120-, 100-, and 50-bp labeled DNA fragments. The origin of the pulse-labeled DNAs is discussed.