and related this to altered mitochondrial DNA replication found in vivo. 5 We explored enzymologic events in the interaction of purified DNA pol-y with AZTTP. Toxic mechanisms in AZT mitochondrial myopathy may be analogous in some ways to pharmacologic mechanisms in the antiretroviral action of AZT.2 The present study showed that AZTTP inhibited DNA pol-y with mixed inhibition kinetics. AZTTP acted both as an alternate substrate for dTTP with DNA pol-y and competed with dTTP for DNA pol-y nucleotide binding (as seen with AZTTP and some cellular polymerases). Materials and Methods MaterialsReagents were analytic grade I. AZTTP was from Moravek Biochemicals, Brea, Calif. [3H]dTTP was from Amersham. All enzyme inhibitor assays were replicated from five to eight times, and each assay point was performed in triplicate within each run. Calculated arithmetic means from the triplicate assays were used to plot kinetic data. Mitochondrial Isolation ProceduresFor mitochondrial isolation, all procedures took place either on ice or at 4°C and resembled those used by us and by others in the past.78 Fresh bovine hearts were obtained from the slaughterhouse (courtesy of Kluener Packing Co, Cincinnati, Ohio) approximately 20 minutes before beginning the preparation. Tissue was minced into 1-cm cubes and homogenized in buffer M that consisted of (mmol/L) sucrose, 250; Tris-HCI, 50 (pH 8.0); 2-mercaptoethanol (2-ME), 5; EDTA, 1; MgCl2, 5; and KCI, 25. Buffer volume was increased 10-fold, and the mixture was homogenized with a hand-held Potter-Elvejhem homogenizer (Fisher Scientific, Pittsburgh, Pa). Debris was pelleted at 500g. The mitochondria were pelleted from the supernatant solution by centrifugation at 8500g for 10 minutes.

The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.

An Escherichia coli mutant, temperaturesensitive for DNA synthesis in vivo and in vitro, is defective in single-strand binding protein (SSB; DNA-binding protein). Conversion of phage G4 single strands to the duplex form is defective in crude enzyme fractions of the mutant and is complemented by pure wild-type SSB. Radioimmunoassays of mutant extracts show normal levels of material crossreacting with anti-SSB antibody. SSB purified to homogeneity from the mutant is active, with lower specific activity, in the reconstituted G4 replication assay at 30'C, but virtually inactive at 420C. Surprisingly, the mutant protein, like the wild-type protein, survives heating at 1000C. Thus, mutant SSB is structurally heat-resistant but is functionally thermosensitive in vitro and in vivo. Both the in vivo and in vitro defects are tightly linked in transductions by phage P1. SSB is required for the conversion, in vitro, of single-stranded phage DNA to the duplex replicative form (3) and in the conversion of the latter to single-stranded viral circles (5). Despite this dependence on SSB for phage DNA replication in vitro, it was not clear whether replication in vivo had a similar requirement. For lack of a mutant defective in SSB, or a specific inhibitor of it, the question of an essential or auxiliary role in replication, repair, and recombination remained in doubt.We report here that SSB is an essential component in phage and cellular DNA metabolism in vivo. A mutant has been identified with a temperature-sensitive defect in SSB. DNA synthesis stops immediately when the temperature is raised from 30'C to 420C. Single-stranded phage (ST-1) production is also blocked at 420C. The isolated mutant SSB shows a similar temperature sensitivity in an in vitro phage DNA replication system. Additional descriptions of the biochemical and genetic features of SSB will be reported elsewhere. MATERIALS AND METHODSE. coli strains were obtained as follows: strain SG1635 (thy A3, dnaM710, ssb-1) and parent DG17 (thyA3) (7) from D. Glaser (University of California, Berkeley, CA), strain KY2750 (dnaP18) and its parent KY2053 (8) from T. Yura (Kyoto University, Kyoto, Japan), and strains AB1157 (9) and AN385 (ubiA -, gal-, strR, X +) (10) from G. Weinstock of this department. SG1635 contains two mutations resulting in temperature-sensitive cell growth. One mutation, originally designated dnaM710, maps at about 75 min on the E. coil genetic map (ref. 7; unpublished results). This mutation is not responsible for temperature-sensitive DNA replication, has no effect on SSB (unpublished results), and thus is irrelevant to the results reported here. The effects of the other mutation (ssb-1) are the subject of this paper. The origins of strains JGC155 and JGC158 are described in this paper.A set of Hfr strains was obtained from B. Bachmann (E. col' Genetic Stock Center, Yale University, New Haven, CT). Hfr mapping was performed as described (11).Complementation assays were carried out in 25-Al reaction mixtures containing 20 mM Tris-HCl (pH 7.5)...

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Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.