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The ability of three different DNA polymerase I mutants of Escherichia coli to carry out excision repair was examined. Strains having the same genetic origin but carrying either the polA1, polA107, resA1 or pol+ alleles were compared. The rate of ultraviolet-induced dimer excision was slightly reduced, relative to that found in Pol+ strains, in the PolA1 strain; greatly reduced in the PolA107 strain; and found not to occur in the ResA1 strain. Ultraviolet light-induced repair synthesis as determined by the ultravioletstimulated incorporation of 3H-labelled 5-bromo-2′-deoxyuridine into DNA of the parental density showed that the polA1 mutation results in an increase in repair replication while the presence of the polA107 allele caused a reduction in the amount of repair synthesis relative to that of the Pol+ strain. The ResA1 strain, however, showed no ultraviolet stimulation of the incorporation of the density label. This observation indicates that DNA polymerase I plays a key role in the excision repair process in E. coli.

Bacteriophage ST-1 replication requires DNA polymerase III ( dnaE ) but not DNA polymerases I or II, DNA ligase, or the products of dnaA, B, or C-D . It was not certain whether dnaG was required. These results differ considerably from those reported for φX-174.

A mutation in gene 49 of phage T4 was not able to restore DNA synthesis in a gene 46 mutant.

IN previous papers it has been shown that the in vitro template activity of DNA1 and chromatin2 with DNA polymerase I of Escherichia coli is altered by the action of ionising radiation. These changes have been attributed1 to the formation of new binding sites on the DNA for the enzyme, some sites being active in the synthesis of DNA and others inactive. These new sites are associated with single-strand breaks in the DNA. By working under conditions either of enzyme excess or of excess of template it is possible to distinguish between newly formed active and inactive enzyme binding sites.

The experiments described in this paper implicate chloroplast protein synthesis in the regulation of nuclear DNA replication. The inhibition of nuclear DNA replication in the lower eukaryote, Chlamydomonas reinhardi strain 21gr , was examined after growth of cells with a series of antibiotics (streptomycin, neamine, spectinomycin, cleocin, chloramphenicol, and rifampicin) each of which has a known effect upon chloroplast RNA or protein synthesis in this organism. Each antibiotic inhibited nuclear DNA replication at drug concentrations at which there was little or no inhibition of adenine incorporation into chloroplast DNA. That chloroplast DNA was replicating under these conditions rather than merely being repaired, was shown first by the high incorporation rates and second by a 14 N- 15 N density transfer experiment in which chloroplast DNA doubled in the presence of streptomycin, while no incorporation into nuclear DNA was detected. A small DNA peak, Component III, located between nuclear and chloroplast DNA's in CsCl gradients, possibly mitochondrial, was more pronounced in DNA from antibiotic-inhibited cultures than from controls.

In vivo , ribosomal RNA of Saccharomyces cerevisiae is transcribed from the light strand of γ DNA. In vitro , γ DNA is transcribed with equal efficiency by both of the yeast nuclear RNA polymerases, polymerase I and polymerase II; however the RNA products synthesized by the two enzymes differ from each other both in size and in the relative composition of guanine and cytosine. RNA synthesized by polymerase I hybridizes preferentially to the light strand, while that synthesized by polymerase II hybridizes equally well to either strand. Selective transcription of the light strand of the ribosomal DNA also occurs when high-molecular-weight total nuclear DNA is used as template.

An enzymatic activity that terminally cross-links T7 DNA has been detected in extracts from phage T7-infected Escherichia coli cells. This activity required the functional products of gene 1.3 (T7 DNA ligase), gene 5 (T7 DNA polymerase), and gene 6 (T7 exonuclease) as well as Mg2+ ions, the four deoxytriphosphates, and ATP. The cross-linking system could be reconstituted with purified ligase, polymerase, and exonuclease, and maximal activity required the presence of all three enzymes. The system was active on phage DNA which possessed unique termini but not on DNA with non-unique ends.

Bacteriophage φX174 DNA replication was examined in temperature-sensitive dnaB mutants of Escherichia coli C to determine which stages require the participation of the product of this host gene. The conversion of the infecting phage single-stranded DNA to the double-stranded replicative form (parental RF synthesis) is completely inhibited at the nonpermissive temperature (41 C) in two of the three dnaB mutants tested. The efficiency of phage eclipse and of phage DNA penetration of these mutant host cells at 41 C is the same as that of the parent host strain. The defect is most likely in the synthesis of the complementary strand DNA. The semiconservative replication of the double-stranded replicative form DNA (RF replication) is inhibited in all three host mutants after shifting from 30 to 41 C. Late in infection, the rate of progeny single-stranded phage DNA synthesis increases following shifts from 30 to 41 C. Approximately the same amounts of phage DNA and of infectious phage particles are made following the shift to 41 C as in the control left at 30 C. The simplest interpretation of our data is that the product of the host dnaB gene is required for φX174 parental RF synthesis and RF replication, but is not directly involved in phage single-stranded DNA synthesis once it has begun. The possible significance of the synthesis of parental RF DNA at 41 C in one of the three mutants is discussed.

We propose a model to investigate the relation between insertion and excision activities of polymerases involved in DNA synthesis, and the frequency of errors resulting from substituting either mismatched bases or base analogues into a DNA molecule. An analytical equation is derived which expresses the error frequency as a function of nucleotide insertion and removal rates. For the general case, given arbitrary rates of insertion and removal, and allowing the enzyme to peel back by excising previously incorporated nucleotides, we have developed a computer simulation for the synthesis of a DNA molecule. In the special case, where insertion and removal frequencies are within the biologically interesting range for spontaneous mutations, the effect of “peelback” on error correction can be obtained analytically. Our results suggest that the magnitude of the removal frequency (3′-exonuclease activity) is the parameter that exerts the greatest influence on error correction capability; the frequency of errors is less sensitive to either the specificity for removal of mismatched relative to correctly matched bases, or to peelback.

The replication of the bacterial plasmids is dependent on two chromosome-determined products (dnaA and dnaC), which are involved in the initiation of the chromosomal DNA synthesis of Escherichia coli. The inhibition of Col E1 DNA synthesis in a dnaA-mutant (CRT 46) at the restrictive temperature, is eliminated by the addition of low doses of chloramphenicol (3 μg/ml). Under these conditions, a stimulated and relaxed synthesis of this plasmid is observed. The ability of chloramphenicol to overcome the inhibition of Col E1 DNA synthesis is dependent on the extent of the residual plasmid DNA synthesis at the time of addition of the antibiotic. Col E1 DNA synthesized at 43 °C in the presence of chloramphenicol, largely consists of supercoiled DNA circles containing RNA segments as revealed by their sensitivity to ribonuclease H of Escherichia coli and alkali. Addition of rifampicin to chloramphenicol-treated cells causes first a slight inhibition of the relaxed Col E1 DNA synthesis at 43 °C and then a further stimulation. An immediate increase in plasmid DNA synthesis after the addition of rifampicin to chlor-amphenicol-treated cells at 43 °C is observed in the dnaA-mutant carrying the antibiotic resistance factor R1. Another transmissible plasmid (Hly PM152) does not show this stimulated DNA synthesis. Stimulation of Col E1 and R1 DNA synthesis by rifampicin does not occur in chlor-amphenicol-treated cells of the dnaA-mutant with a rifampicin-resistant RNA polymerase. Chloramphenicol and/or rifampicin cannot eliminate the inhibition of plasmid replication at the restrictive temperature in a dnaC mutant (PC 2). In the presence of chloramphenicol, however, more complex Col E1 DNA molecules (dimeric and trimeric DNA forms) are synthesized at the restrictive temperature in the dnaC-mutant.

Exonuclease VII from Escherichia coli is specific for single-stranded DNA, degrading denatured DNA, single-stranded regions extending from the ends of duplex DNA, or displaced single-stranded regions. Hydrolysis is initiated at both 3′ and 5′ termini. Exonuclease VII can also excise thymine dimers from duplex DNA following treatment of the DNA with the Micrococcus luteus endonuclease specific for thymine dimers. The enzyme has no detectable activity on RNA or DNA-RNA hybrid molecules. The limit products of exonuclease VII action are oligonucleotides bearing 5′-phosphoryl and 3′-hydroxyl termini predominantly in the range of tetramers to dodecamers; no mononucleotide products have been observed. The enzyme acts in a processive fashion, initially releasing large, acid-insoluble oligonucleotides which can be degraded further to produce a limit product of acid-soluble oligonucleotides. The possible role of exonuclease VII in recombination and in the repair of ultra-violet damage to DNA is discussed.

DNA synthesis in an in vitro system proceeds by a covalent chain extension of the newly generated primers in the presence of Mg2+, Escherichia coli DNA polymerase I or its large fragment, ribo- and deoxyribonucleoside triphosphates, RNA polymerase, and either single-stranded fd DNA or d(T)1000 as template. The synthesis is strongly influenced by the number of RNA chains and the kinetics of polymerization is nonlinear, showing a brief lag followed by a period of rapid synthesis before reaching a saturation level. However, a linear kinetics is observed when the number of new chain initiation is controlled (i.e. rifampicin added 1 to 5 min after RNA synthesis). A degradation of ribo-strands is observed during DNA synthesis. When d(T)1000 fully transcribed with RNA polymerase (1:1 hybrid) is employed as template-primer, both the whole enzyme and the large fragment of DNA polymerase I catalyze incorporation of dAMP residues. During the initial synthetic period there is a mole for mole degradation of ribo-strands by the whole enzyme. With large fragment DNA chain growth proceeds by a process of of strand displacement. In the initial synthetic period the 5'→3' exonuclease activity of DNA polymerase I attacks the 5' ends of the ribostrands endonucleolytically liberating 5'-triphosphate-terminated di- or oligonucleotides. Subsequent action of the nuclease activity proceeds exonucleolytically liberating 5'-mononucleotides as the DNA chain growth proceeds. The results suggest that the 5'→3' exonuclease activity of DNA polymerase is capable of removing RNA primers from the final product of replication in vivo.

Addition of Carbopol 934 (carboxypolymethylene, carboxyvinyl polymer) to an in vitro RNA-dependent DNA polymerase assay system resulted in a significant increase in the rate and extent of DNA synthesis. Carbopol may be a valuable tool for increasing the sensitivity of detection of reverse transcriptase activity in oncogenic RNA viruses and malignant tissues.

We have previously reported that an RNA polymerase mutation, rif R 2, of Escherichia coli retards T4 bacteriophage development. The effect of rif R 2 on phage production is partially suppressed in β-glucosyl transferaseless mutants of T4 and β-glucosylation of the parental DNA alone is sufficient to inhibit phage growth. Both physiological studies and the map positions of other suppressing T4 mutations (in genes 45 and maybe 55) support the conclusion that there are two effects of rif R 2 during phage development: a direct effect on T4 DNA replication and a direct effect on late gene expression. Both of these defects seem in part to be due to a failure of the mutant RNA polymerase to perform a reaction(s) which physically alters or changes the association of parental and then, probably, some progeny DNA. This reaction is required early for T4 DNA replication and throughout infection for late gene expression.

We have compared the relative inhibitory activity of several synthetic and natural ribonucleic acids against four cellular and two viral DNA polymerases. We have found that except for poly(rA)-dependent DNA polymerase (which copies poly(rA) strand of poly(rA) · (dT)10 in the presence of Mn2+ at an efficiency higher than that of any template) all other human cell polymerases tested (3–4-S) DNA polymerase, 6–7-S DNA polymerase, and terminal deoxynucleotidyl transferase) either were not inhibited by poly(A), poly(C), poly(U), tRNA and ribosomal RNA or were inhibited only at very high concentrations of these agents compared to those required for inhibition of endogenous RNA-directed DNA polymerase from murine leukemia virus (MLV) and feline leukemia virus (FeLV). Furthermore, following thiolation, poly(C) and ribosomal RNA were potent inhibitors while still retaining their selectivity. These results show that natural and synthetic polynucleotides were far more inhibitory to viral enzymes than to cellular enzymes, except for poly(rA)-dependent DNA polymerase.

Pretreatment of native DNA with nucleases which hydrolyze phosphodiester bonds with liberation of 3′-hydroxyl terminal groups is known to increase the rate of incorporation of nucleotides into DNA by E. coli DNA polymerase I and similar enzymes. Concentration ranges and conditions for this reaction have been established which allow specific detection and quantitative assay of such nucleases. The method permits detection of as little as 0.1 pg of pancreatic deoxyribonuclease, is simple, and requires only commercially available components. It has been utilized to monitor purification of a nuclease from HeLa cell nuclei at concentrations which could not be detected by other methods.

The effect of different inhibitors of DNA synthesis on the labeling of mitochondrial DNA, isolated by CsCl-ethidium bromide density gradient and sedimentation velocity, has been studied in HeLa cells. Thymidine and deoxyadenosine excess, hydroxyurea and fluorodeoxyuridine inhibit the labeling of mitochondrial DNA less than that of the nuclear DNA (with a maximum difference of 22%) while cytosine arabinoside, which strongly inhibits nuclear DNA synthesis, has a very limited action on mitochondrial DNA, in analogy to what happens in the bacterial system. Ethidium bromide inhibits, as expected, only mitochondrial DNA replication.

The E. coli DNA binding protein reduces the activity of the single-strand-specific nucleases associated with all three DNA polymerases known in E. coli . A slight excess of binding protein over that required to saturate the DNA template leads to total inhibition of activity of the 3′ → 5′ nucleases associated with DNA polymerases I and III, but restores maximum activity of the DNA polymerase II-associated nuclease. The binding protein forms a specific complex with DNA polymerase II in the absence of DNA, and it is this complex that degrades a DNA·binding protein complex. Binding protein also facilitates the binding of DNA polymerase II to single-stranded DNA, whereas the binding to DNA of DNA polymerase I is inhibited. These data may explain the specificity with which the binding protein enhances the synthetic ability of DNA polymerase II.

Certain enzymatic properties of homogenous DNA polymerase I from Acinetobacter calcoaceticus have been investigated. The enzyme requires an initiator DNA for normal DNA repair synthesis. It is, however, also capable of de novo synthesis in the presence of dATP and dTTP or dGTP and dCTP. The enzyme was only partially inhibited by the–SH-group-blocking reagent p-hydroxymercuribenzoate. DNA polymerase I from Acinetobacter calcoaceticus contained both a 3′ 5′ and a 5′ 3′ exonuclease activity. The products of the former was 5′-deoxymononucleotide phosphates. The 5′ 3′ nuclease gave mononucleotides with T7 DNA and with d(A-T)n the products were mono, di, and trinucleotides, the ratio depending on the concentration of Mg2+· The 3′ 5′ exonuclease activity appeared to be slightly more active on single-stranded than double-stranded DNA. The opposite was the case for the 5′ 3′ nuclease which had an absolute requirement for double-stranded DNA. The present work offers further evidence for the view that DNA polymerase I enzymes in microorganisms have similar structural features and enzymatic properties.

Earlier studies showed that the M13 single-stranded circle is converted to duplex replicative forms (RF) in vivo and in vitro by a rifampicin-sensitive system that fails to act on φX174 DNA. In the absence of DNA polymerase I, the synthetic complementary strand produced by a crude cell extract contained a small gap at a unique position relative to the viral template strand. In these studies we show that the conversion of viral M13 DNA to the covalently closed duplex form requires the action of six purified proteins: RNA polymerase, DNA unwinding protein of Escherichia coli, DNA polymerase III*, copolymerase III*, DNA polymerase I, and DNA ligase. In the absence of polymerase I, the location of the gap in the RF produced by the reconstituted protein system is the same as that produced by the crude cell extract. The functions of the several proteins, either demonstrated or inferred, are as follows: RNA polymerase to form a priming fragment, DNA unwinding protein to mask the single-stranded DNA in all but the promoter region for RNA polymerase and to support polymerase III*-copolymerase III* in carrying out most of the DNA chain growth, polymerase I to fill the small remaining gap, including that created by its 5'→3' exonucleolytic excision of the remaining RNA fragment, and ligase to join the polynucleotide ends to complete the synthetic complementary circle. Essential functions of RNA polymerase, polymerase III*, and polymerase I in the crude system have been verified by studies of extracts of strains deficient in these proteins. The DNA unwinding protein coded by gene 5 of M13 and essential for synthesis of the viral strands later in infection, fails to replace the E. coli unwinding protein, but instead inhibits conversion of the single strand to RF.

We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA(+) toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe(12) and argA(3) genes.

A 2 hr-incubation of gamma-irradiated E. coli polA1 tsdnaE at 45° prior to plating at 30° results in the decrease of the fraction of viable bacteria. This effect is not observed in experiments with gamma-ray sensitive polA1 dnaE+ and gamma-ray resistant polA1+ tsdnaE bacteria. The study of uptake of H3-thymine or H3-uridine into DNA or RNA of polA1 tsdnaE and polA1 dnaE+ cells shows that the decrease in survival at 45° in the first strain after gamma-irradiation correlates with the DNA synthesis block rather than with the unspecific inhibition of RNA synthesis. DNA breakdown in gamma-irradiated polA1 tsdnaE cells was found to be higher at 45° as compared with 30°. The results may be interpreted as indicative of the participation of DNA-polymerase III in the repair of gamma-ray induced lesions possibly during replication.

Summary E. coli RNA polymerase-binding sites on fl RFI DNA were isolated as DNA fraction (1.9% of total DNA) protected by RNA polymerase against the nucleolytic digestion. These polymerase-protected DNAs had the properties as follows:(1) They were not obtained in the absence of sigma factor. (2) They were short double-stranded DNA fragments(35–40 base pairs long) and enriched in A-T base pairs (65%). (3) After heatdenaturation, they exhibited the resistance to the single strandspecific nuclease to some extent, but the nuclease-resistance was very temperature dependent. They behave not like GC-rich regions on fl DNA, which have very stable hairpin-like structure.

Exonuclease V ( recBC DNase) is inactivated in E. coli between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.

Semi-conservative DNA synthesis is observed when the isolated, folded E. coli chromosome is supplemented with a DNA-free, soluble enzyme fraction, the four deoxynucleoside 5′-triphosphates, ATP, and Mg ++ . The DNA synthesized in vitro remains associated with the folded chromosome during sedimentation through neutral sucrose, but is released as small DNA fragments in alkali. Sealing of these replicative intermediates to the chromosome requires the presence of both E. coli DNA polymerase I (EC 2.7.7.7) and DNA ligase (EC 6.5.1.2).

Replicative form DNA composed of a closed complementary strand and a discontinuous viral strand has been isolated from cells infected with bacteriophage φX174 during the period of single-strand DNA synthesis. This RFII DNA was degraded by the restriction enzyme from Hemophilus influenzae, endonuclease R, and the products analyzed by polyacrylamide gel electrophoresis. The results indicate that there are two types of discontinuity in the viral strands of these molecules: (1) 65% of the molecules contain a gap, which causes a discrete increase in mobility of a specific restriction enzyme fragment, R3. This gap can be selectively repaired with Escherichia coli DNA polymerase I and nucleoside triphosphates, but the molecules are not converted to RFI by addition of E. coli polynueleotide ligase to the reaction mixture. Approximately 30 moles of radioactive TTP are incorporated per mole of RF DNA. (2) 35% of the RF molecules contain a discontinuity, which does not result in a detectable change in mobility of any restriction enzyme fragment. These RF molecules can be converted to RFI by the action of ligase and polymerase I in the presence of nucleoside triphosphates, with incorporation of only approximately one mole of radioactive TTP, specifically into fragment R3, per mole of RF DNA. When the reaction of late RFII DNA and polymerase I is allowed to proceed beyond the repair of the discontinuity, radioactive nucleotides are incorporated into endonuclease R fragments adjacent to R3 in the 5′ → 3′ direction. This technique was utilized to determine a partial order of endonuclease R fragments in φX174. These results suggest that the synthesis of single-strand DNA is initiated from a unique point in cistron A and proceeds clockwise round the φX174 genetic map (cistron order: ABCDEFGH). A comparison of these results with other studies on φX174 suggests that DNA synthesis in all stages of φX174 replication may be initiated from a specific locus on the genome, at or near cistron A.

A cell-free system for the study of bacteriophage T7 DNA replication has been developed. DNA synthesis in vitro requires the four deoxynucleoside triphosphates and an exogenous T7 DNA template. The rate of synthesis is increased 5- to 6-fold by the four ribonucleoside triphosphates. The products of the phage gene 4 and gene 5 (DNA polymerase), but not those of genes 2, 3, and 6, are required for DNA synthesis in vitro. The kinetics of synthesis is linear for 20 to 30 min, and 30 min after the start of the reaction the amount of DNA synthesized is 2 to 3 times the amount of template DNA added to the reaction. The product is not covalently attached to the template. Analysis by zone sedimentation through alkaline sucrose gradients indicates that the products of synthesis are short (11 S) DNA chains, suggesting that the sealing of "Okazaki pieces" into DNA of high molecular weight is inefficient in this in vitro reaction. Examination of the product in the electron microscope reveals many large, forked molecules, indicating that in some cases large portions of the T7 genome are replicated in vitro.

The net DNA synthesis that persists at the restrictive temperature in the conditional lethal DNA ligase mutant Escherichia coli lig ts7 is semiconservative, suggesting that although the rate of joining of 10 S “Okazaki fragments” in the mutant is greatly reduced, it is nevertheless sufficient to permit continued progression of the replication fork and the initiation of new rounds of replication. DNA synthesis in E. coli lig ts7 of a phage (P2) that replicates its chromosomes unidirectionally is discontinuous on both strands. A double mutant of E. coli K12, ( lig ts7 pol A12), has been constructed which bears a temperature-sensitive mutation ( pol A12) in the structural gene for DNA polymerase I in addition to the lig ts7 mutation. Joining of the Okazaki fragments in the double mutant occurs at a slower rate than in either the lig ts7 or pol A12 parents. In contrast to the behavior of the single mutants, DNA synthesis in the double mutant stops abruptly upon shift from 25 °C to 42 °C.

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90 S as compared to 34 S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34 S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90 S viral DNA represents adenovirus DNA covalently integrated into cell DNA.

Near-ultraviolet photoproducts of l -tryptophan (TP) differentially inhibited deoxyribonucleic acid (DNA) replication in wild-type cells and uvrA, polA1 , and recA recB double mutants of Escherichia coli . Wild-type cells labeled in their DNA with [ 3 H]thymidine in the presence of TP produced small pieces of DNA (7 × 10 6 daltons), which corresponded in size to late replicative intermediates of discontinuous DNA synthesis. Moreover, when TP was present, it took five times longer to chase the low-molecular-weight DNA pieces into high-molecular-weight DNA. The observation of replicative intermediates in the presence of TP, and their slow chase into high-molecular-weight DNA in the presence of TP, is strong evidence that TP stabilizes replication gaps in E. coli DNA. Although TP slowed DNA replication in wild-type cells, this effect was transient and DNA synthesis eventually resumed at a normal rate. However, in polA1 and recA recB mutants, DNA synthesis was completely inhibited. Determinations of size and total counts of cells incubated in TP suggested that TP uncouples cell division from DNA replication in recA recB mutants, whereas these processes remain coupled in wild-type cells and in uvrA and polA1 mutants. The slow chase of TP-stabilized pieces of DNA in the presence of TP suggested that the selective effect of TP on DNA synthesis and viability in repair-deficient mutants is a result of TP inhibition of replication gap closure.

Reconstitution of Escherichia coli RNA polymerase was found to be markedly enhanced by DNA as well as by the σ subunit. Among discrete steps of subunit assembly, formation of the primary intermediate α 2 β complex and subsequent association of the complex with the β′ subunit are not affected by the presence of DNA and the σ subunit; the α 2 ββ′ complex thus formed, however, is virtually inactive and is subject to temperature-dependent activation by DNA and the σ subunit. The α 2 ββ′ complex is, therefore, a secondary intermediate in the sequence of enzyme formation, or a premature form of core enzyme. In the course of activation of the premature core complex, the subunit σ interacts with both the α 2 β complex and the β′ subunit; DNA acts in much the same way. The enzyme, reconstituted in the presence of DNA, is recovered attached to the DNA, added as an enhancer, and initiates RNA synthesis without prior release from the DNA. A limited number of unique DNA sites appear to be concerned with the enzyme maturation.

DNA synthesis has been studied in T4-infected Escherichia coli cells made permeable to nucleotides by treatment with toluene. The rate of incorporation of labeled deoxyribonucleoside triphosphates into DNA at various times after infection is proportional to the in vivo rate. This in vitro incorporation is dependent on all four deoxyribonucleoside triphosphates (5-hydroxymethyldeoxy-cytidine triphosphate can substitute for dCTP) and Mg 2+ . It is stimulated by rATP, partially inhibited by pancreatic DNase, and abolished by N -ethylmalei-mide and 1-β- d -arabinofuranosylcytosine triphosphate. T4 amber DO (DNA negative) and temperature-sensitive DO mutants under nonpermissive conditions of infection fail to induce DNA synthesis in vitro. The synthesizing activity is intracellular and the DNA product is exclusively T4 DNA. The in vitro synthesis proceeds in a discontinuous manner involving synthesis and subsequent joining of small DNA fragments (about 10 S in alkaline sucrose gradients) into larger molecules predominantly one-half the length of mature T4 DNA. No restriction of C-containing or nonglucosylated HMC-containing T4 DNA product is observed in this system.

The localization and properties of DNA polymerases I and II are reviewed with particular regard to changing activities during rat liver regeneration and in hepatomas. DNA polymerase I is normally found in the nucleus and is also associated with ribosomes, from which it can be eluted and purified. This enzyme does not vary as a function of growth rate or malignancy. DNA polymerase II is normally associated with post-microsomal membrane fragments. During periods of rapid growth its activity rises markedly in both membranes and nuclei. It has been reported from this laboratory that this enzyme exists as a complex together with thymidine kinase, thymidylate synthetase and ribonucleotide reductase. We have speculated that this may be part of a replicative unit for DNA biosynthesis. The selective inhibition of DNA polymerase II by nitrosoureas and their isocyanates is of considerable interest. It is proposed that certain other chemotherapeutic agents may possibly act via a similar type of inhibition of this or other enzymes in this multienzyme complex and that this may also be a fruitful approach in searching for new agents.

The ultraviolet inactivation of DNA polymerase I and II both followed first order kinetics. There was no alteration in enzyme properties such as optimum concentration of magnesium chloride or of DNA template-primer, pH profile, or substrate saturation curve, and hence is compatible with an all-or-none effect. The rate of ultraviolet inactivation of DNA polymerase II was about 3 times that of DNA polymerase I, and this difference was further augmented when 5-iodo-2'-deoxyuridine 5'-triphosphate (IdUTP), a metabolic analog of deoxythymidine triphosphate, was included during irradiation. The enhanced ultraviolet inactivation observed in the presence of IdUTP of both DNA polymerase I and II was not only dependent on the IdUTP concentration but also was a saturable phenomenon. 5-Iodo-2'-deoxyuridine and sodium iodide sensitized these enzymes to ultraviolet radiations; however, the enhanced rate of inactivation by ultraviolet light was linear over the concentrations used. Magnesium chloride by itself had no effect on the inactivation of either DNA polymerase I or II; however, magnesium chloride further enhanced the sensitizing effect of IdUTP and of 5-iodo-2'-deoxyuridine but not of sodium iodide. The optimum magnesium chloride concentration for such an effect was identical with that required for optimum catalytic activity. The molar ratio of magnesium chloride to IdUTP was not crucial for this effect. The DNA template also sensitized the polymerases toward inactivation by ultraviolet irradiation, the effect being greater with DNA polymerase II relative to DNA polymerase I. DNA further increased the IdUTP-sensitizing effect to ultraviolet radiation to the same extent as magnesium chloride. The free radical intermediate postulation proposed previously (Cysyk, R., and Prusoff, W. H. (1972) J. Biol. Chem. 247, 2522) for the 5-iodo-2'-deoxyuridine ultraviolet inactivation of thymidine kinase was also considered for the present IdUTP-DNA polymerase system.

T4 DNA polymerase cannot use a nicked duplex DNA molecule as a template-primer for DNA synthesis, apparently because it is unable to displace the 5' end of the strand paired to the strand serving as the template. Addition of the T4 phage gene 32 DNA unwinding protein (32-protein) facilities strand displacement and allows the T4 DNA polymerase to synthesize DNA using nicked duplex T7 DNA as the template-primer. The first product of this reaction is a rapidly renaturable DNA which is found to contain a few large branches when examined by electron microscopy. Later in the reaction there is also synthesis of a product containing predominantly alternating residues of dAMP and dTMP.

The newly synthesized DNA after UV-irradiation of uvr+ or uvrE. coli cells has a low single-stranded molecular weight equal to the average spacing between pyrimidine dimers in parental strands of DNA [ 1,2] . The decrease of DNA molecular weight in alkaline sucrose gradients is caused by the gaps (1000 nucleotides long) in daughter strands, opposite to the pyrimidine dimers, arising at the replication fork [ 1,3]. In the course of subsequent incubation in growth medium these gaps are repaired. The finding of DNA strands with intermediate density in alkaline CsCl in UV-irradiated E. coli uvrA6 cells allowed the authors to suggest that post-replication gaps are repaired by a recombination mechanism involving actual breakage and reunion of DNA molecules [4]. However, many facts do not accord with this hypothesis [5,6]. For example, E. coli AB 2487 recAI3 cells show no deficiency in post-replication repair after 15 erg/mm? of 254 nm W/-irradiation although the relative recombination frequency in these cells is about 10T4 compared to a value of 1 for ret+ cells [7] ; on the other hand, there is no breakage of parental DNA strands in UV-irradiated uvrA6 or uvrB5 cells at all [7,8]. It is possible therefore that UV-stimulated recombination between sister DNA duplexes, if it occurs, does not causally relate to the filling of gaps and the latter are repaired by de novo DNA synthesis [5 ] . In this paper data are presented indicating that inactivation at the non-permissive temperature (45’CO of thermolabile DNA polymerase III in a double polAI p0lC26~~ mutant of E. coli results in the failure to repair post-replication gaps after W-irradiation.

Arylhydrazinopyrimidines inhibit DNA synthesis in Bacillus subtilis by promoting formation of a specific, long-lived ternary complex with DNA polymerase III and the template-primer DNA. DNA polymerase III contains an associated, single-strand-selective exonuclease which generates 5′-mononucleotides. Drug inhibition of the nuclease similarly proceeds through formation of the ternary complex. The ternary complex was isolated by agarose chromatography. Like inhibition of the nuclease, optimum formation of the complex requires duplex DNA with single-stranded regions such as bacteriophage λ DNA (purely single- and double-stranded DNA are ineffective) and is antagonized by specific deoxyribonucleoside triphosphates. Formation of the ternary complex requires a di- or polyvalent cation and is inhibited by sulfhydryl reagents and high ionic strength. The complex dissociates with a half-life of the order of minutes at 4°.

The replication of the bacteriocinogenic factor Clo DF13 was studied in Escherichia coli mutants which lack either DNA polymerase I (polA1 and resA1 mutants), DNA polymerase II (polB1 mutant) or DNA polymerase III (dnaE mutant). DNA polymerase I is required for Clo DF13 replication. The Clo DF13 factor, however, can be maintained in a strain carrying the polA107 mutation and thus lacking the 5′→3′ exonucleolytic activity of DNA polymerase I. DNA polymerase II is not required for transfer replication and maintenance of the Clo DF13 plasmid. In the temperature sensitive dnaE mutant, Clo DF13 can replicate at the nonpermissive temperature during the first two hours after the temperature shift from 30°C to 43°C. During this period DNA polymerase III seems not to be essential for Clo DF13 replication.

DNA-dependent RNA polymerase was easily solubilized from the apical part of cauliflower inflorescences into homogenizing medium containing ammonium sulfate. A distinct RNA polymeraseshave been isolated by DEAE-cellulose column chromatography. DEAE-polymerase II was more effectively primed when denatureded calf-thymus DNA, rather than native DNA, was used as a template. Cauliflower DNA extracted from the inflorescence was more effectively utilized when cauliflower RNA polymerase, rather than calf-thymus DNA, was used as a template for RNA synthesis.

The time course of the appearance of intracellular viral DNA has been studied in mouse L cells infected with the single-stranded DNA virus MVM (minute virus of mice) by using a selective extraction procedure. Approximately half of this DNA elutes from hydroxyapatite as single-stranded DNA. It is sensitive to Escherichia coli exonuclease I and shows a sedimentation profile similar to DNA from the virus, suggesting that it is progeny viral DNA. The remainder of the selectively extracted DNA elutes from hydroxyapatite in the position of double-stranded DNA and is resistant to exonuclease I. Most of this DNA has a sedimentation coefficient of 14 to 16 S , indicating that its molecular weight is twice that of the viral DNA. Denaturation renders the majority of the double-stranded DNA sensitive to exonuclease I, but a significant fraction renatures spontaneously in a monomolecular fashion, indicating that it has a cross-linked or hairpin structure. Chromatography of the double-stranded DNA on benzoylated diethylaminoethyl cellulose resolves two components, one with duplex structure and one which contains single-stranded regions. A short pulse label late in infection predominantly labels the latter class of DNA, suggesting that it contains replicating intermediates. The possible roles of these various forms of DNA in the replication of the viral genome are discussed.

CV1 cells infected with simian virus 40 were treated 27 hr after infection with inhibitors of DNA synthesis, and the fate of replicating viral DNA molecules was followed. Miracil D, chloroquine, cytosine arabinoside (ara-C) and hydroxyurea (HU) halted SV40 DNA replication within 20–30 min after addition. Miracil D and chloroquine act by preventing initiation of replication, while ara-C and HU prevent elongation of nascent DNA strands. This was demonstrated using the wild-type strain of SV40, as well as the thermosensitive tsA30 mutant of SV40; in the latter case the effect of the inhibitors was examined on the reinitiation of SV40 DNA synthesis after transfer of tsA30 infected cells from nonpermissive to permissive temperature. The inhibitory effect of fluorodeoxyuridine on SV40 DNA synthesis was partial, resulting in the accumulation of SV40 DNA component II molecules. Treatment with ara-C, HU, or fluorodeoxyuridine, had no effect on the rate of transcription of SV40 DNA, showing that continuous transcription of late SV40 genes does not require continuous replication of viral DNA. In contrast, treatment with miracil D or chloroquine resulted in inhibition (70–90%) of the overall rate of transcription of SV40 DNA.

Ten temperature-sensitive mutants of adenovirus 12 defective in viral DNA replication at the nonpermissive temperature (40°) were selected and seven of them were classified into three groups (A, B, and C) by complementation tests in viral DNA synthesis. The mutants in Groups A, B, and C were defective in initiation of viral DNA synthesis at 40°, as revealed by viral DNA chain elongation or ligation and density label in temperature shift-up experiments. Analysis by the M band technique indicated that the mutants in Groups B and C were less defective than the mutant in Group A in formation of viral DNA replication complex at the nonpermissive temperature. Additional differences among mutants in Groups A, B, and C were also shown by temperature shift-down experiments. All these mutants were not defective in formation of T antigen in permissive cells and in induction of cellular DNA synthesis in nonpermissive cells at the nonpermissive temperature. These observations suggest that at least three viral genes are involved at different steps in initiation of viral DNA replication.

DNA replication in Escherichia coli was followed using analytical composite agarose-acrylamide gel electrophoresis of native and denatured DNA samples from cells that had been pulse-labeled in vivo . The results obtained with samples of native DNA, combined with the different sensitivities of the various electrophoretic fractions of native DNA to enzymatic attack, led us to conclude that a significant amount of single-strandedness exists in the region of the replication site. Data obtained from kinetic analyses of the labeling of both the high molecular weight DNA and the fragments formed on denaturation of native DNA samples were consistent with the Okazaki model (Okazaki et al. , 1968 a,b ) of DNA replication. Furthermore, the data indicated that eight to ten precursor fragments are present per fork during bacterial growth at both 20 and 37 °C, under the conditions used in this study.

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO(-) (3))) residues by treatment with bisulfite. The poly(U(SO(-) (3))(2), C(3)) and poly(U(SO(-) (3))(9), C(1)) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO(-) (3)) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO(-) (3)) residues into U.

Double-stranded, covalently closed circular DNA from phage PM2 was either treated with methyl methanesulfonate, irradiated with ultraviolet light, or irradiated with γ-rays to introduce a small number of lesions per DNA molecule. The damaged DNA preparations were then exposed to an endonuclease from calf thymus that catalyzes the formation of single strand interruptions at apurinic sites in double-stranded DNA. DNA containing 2 to 3 alkylated residues per molecule was not attacked by the enzyme. DNA exposed to ultraviolet light was sensitized to the ultraviolet endonuclease from Micrococcus luteus, which attacks DNA containing pyrimidine dimers, but was resistant to the thymus endonuclease. On incubation in solution, alkali-labile sites appeared in the damaged DNA molecules as secondary lesions, and simultaneously the DNA became sensitive to the enzyme. Similar results were obtained with γ-irradiated DNA. The thymus endonuclease apparently is specific for apurinic sites and analogous lesions involving loss of a base residue from DNA.

The abnormal DNA replication of bacteriophage T4 containing amber nonsense mutants in gene 59 (amC5, amHL628) has been studied. Zonal centrifugal analysis of the mutant intracellular DNA molecules isolated from a nonpermissive host, Escherichia coli B, reveals the following: (1) Parental mutant DNA as well as nascent mutant DNA can be recovered by sucrose gradient centrifugation as a fast sedimenting fraction (>1500 S) which is presumably a cell-membrane DNA replication complex. (2) At 2–3 min after initiation of viral DNA synthesis, a gradual accumulation of mutant DNA with a sedimentation coefficient of 63 S is observed. The molecular weight of this DNA is close to, or a little less than, that of a mature viral DNA, suggesting that less than one round of replication occurs before the complete arrest of DNA synthesis. (3) In contrast to what is found in wild-type infected cells, little or no long single-stranded DNA (concatemer) appears in mutant infected cells. Concatenated molecules of DNA, which have been proposed as intermediates in viral replication, are normally found in wild-type infected cells 7 min after infection. (4) Unlike the case of cells infected with mutants of gene 46 and 47, addition of chloramphenicol prevents premature release of the mutant DNA of gene 59 from the fast-sedimenting fraction (>1500 S).