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P Hanawalt

Publications and source records attributed to P Hanawalt.

At least 19 recordsLinked to original sources
PubMed1989

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.

Bacteriophage lambda↗
PubMed1986

Comparative effects of growth inhibitors on DNA replication, DNA repair, and protein synthesis in human epidermal keratinocytes.

Cultured human epidermal keratinocytes were used as a model system for testing compounds with potential therapeutic effect against hyperproliferative skin disorders. We have investigated whether each test compound caused direct damage to the DNA or inhibited DNA repair and/or seminconservative replication of DNA, as well as its effect on the overall rate of protein synthesis and on expression of specific keratin genes. The following compounds were studied: (a) inhibitors of DNA polymerase alpha [aphidicolin and its derivative aphidicolin glycine], (b) inhibitors of topoisomerases [novobiocin, nalidixic acid, teniposide, etoposide, and 4'-(9-acridylamine) methanesulfon-m-anisidide], (c) modifiers of chromatin structure [sodium butyrate, 3-aminobenzamide, and nicotinamide], (d) inhibitors of calmodulin activation and protein kinase C [chlorpromazine and trifluoperazine]; and (e) drugs used in clinical dermatology [anthralin, fluocinolone acetonide, ketoconazole, and hydroxyurea]. The compounds were tested at concentrations at which they were known from the literature to be effective in their respective actions. Among the groups of compounds studied, the topoisomerase inhibitors were particularly interesting since they caused no detectable damage to DNA but exhibited maximal inhibitory effect on replication combined with minimal inhibition of DNA repair. In addition most of the topoisomerase inhibitors, particularly novobiocin, changed the pattern of gene expression by inhibiting the synthesis of certain keratins and inducing a Mr 67,000 protein in the prekeratin fraction. These properties combined with minimal systemic side effects may encourage the clinical exploration of some topoisomerase inhibitors for antiproliferative therapy of skin disorders.

Anthralin↗
PubMed1979

Deoxythymidine sugars are not direct precursors of DNA-thymine.

A theoretical model for the kinetics of uptake of a putative precursor molecule into nucleotide pools and into replicating DNA has been developed. The relationship between the accumulation of radioactively labeled precursors in the pool and the appearance of radioactivity in DNA is then derived. Experiments have been carried out in bacteria to compare the uptake of radioactive thymine into deoxythymidine triphosphate, deoxythymidine diphosphate sugars, and DNA to test the suitability of either compound as the direct precursor of thymine in DNA. New one-dimensional, thin-layer chromatographic procedures were used to determine the specific activity of deoxythymidine triphosphate and deoxythymidine triphosphate and deoxythymidine diphosphate sugars in growing cultures of 32PO4-labeled Escherichia coli during pulse labeling with [3H]-thymine. A comparison of the experimental data with our theoretical model supports the hypothesis that deoxythymidine triphosphate, but not deoxythymidine sugar, is the direct precursor of thymine in normally replicating DNA in vivo.

DNA Replication↗
PubMed1976

T4-endonuclease V-sensitive sites in DNA from ultraviolet-irradiated human cells.

Human diploid cells (WI38) were pre-labeled with 32Pi, exposed to ultraviolet irradiation and then pulse labeled with [3H]thymidine. The extracted DNA from these cells was subsequently treated with the T4-endonuclease V, an enzyme which specifically nicks DNA strands at positions adjacent to pyrimidine dimers. Sedimentation in alkaline sucrose gradients revealed that the DNA synthesized after irradiation, as well as that made before, contained endonuclease-sensitive sites. Our results suggest that pyrimidine dimers are transferred from parental to daughter DNA strands during post-irradiation incubation. Sedimentation in neutral sucrose gradients showed that the molecular weight of native DNA was not affected by the endonuclease treatment, suggesting that the gaps appearing in daughter strands after irradiation are not opposite dimers or that the enzyme cannot recognize dimers in the gap regions.

Cell Line↗
PubMed1976

Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport.

A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine. Thymidine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation.

Adenosine↗
PubMed1975

DNA turnover and strand breaks in Escherichia coli.

The extent of DNA turnover has been measured in a dnaB mutant of Escherichia coli, temperature sensitive for semiconservative DNA replication. At the nonpermissive temperature about 0.02% of the deoxynucleotides in DNA are exchanged per generation period. This turnover rate is markedly depressed in the presence of rifampicin. During thymine starvation strand breaks accumulate in the DNA of E. coli strains that are susceptible to thymineless death. Rifampicin suppresses the appearance of these breaks, consistent with our hypothesis that transcription may be accompanied by repairable single-strand breaks in DNA. DNA turnover is enhanced severalfold in strands containing 5-bromodeoxy-uridine in place of thymidine, possible because the analog (or the deoxyuridine, following debromination) is sometimes recognized and excised.

DNA Nucleotidyltransferases↗
PubMed1975

Sedimentation analysis of deoxyribonucleic acid from thymine-starved Escherichia coli.

During thymine starvation, strand breaks accumulate in the chromosomal deoxyribonucleic acid (DNA) of Escherichia coli. This effect occurs to a varying extent in different strains and is particularly enhanced in strains deficient in DNA polymerase I. The inhibition of ribonucleic acid or protein synthesis suppresses the accumulation of strand breaks. In a polA strain, rifampin is more effective than chloramphenicol or puromycin in suppressing strand break accumulation. To a certain extent the pehenomenon othymineless death correlates with the appearance of strand breaks. Although the killing can not be explained by the bulk of strand breaks, it is possible that some of them represent lethal events. On the basis of our observations we proposed the following model. (i) Transcription may be accompanied by single-strand breaks in DNA. (ii) DNA polymerase I is involved in the efficient repair of these breaks. (iii) Thymine deprivation results in the accumulation of unrepaired breaks. (iv) Polymerase I-mediated repair is less affected by thymine deprivation than are the alternative pathways because it closes the breaks with short patches, requiring less thymine.

Cell Survival↗