Our broad objective is to understand molecular mechanisms for replication, processing, and repair of DNA in bacteria and in human cells. Our studies focus principally upon cellular responses to treatments that damage DNA and/or inhibit DNA replication. This research is relevant to an understanding of the molecular basis of hereditary diseases, such as xeroderma pigmentosum, in which DNA repair is defective. I. DNA repair is being studied in permeabilized cells from UV irradiated XP and WI38 fibroblasts, in which we have shown phage T4 endonuclease V stimulation of repair synthesis. Kinetics of this synthesis, correlation with pyrimidine dimer excision and effects of other added nucleases are being examined. The action of cytosine arabinoside on DNA repair is being examined. Repair in human cells containing photoadducts of 8-methoxypsoralen or angelicin is under investigation. II. DNA replication inhibition in E. coli by nalidixic acid, coumermycin, thymine starvation, UV irradiation, and dnaB at 42 degrees is being studied, with particular emphasis on the possible involvement of 3'-5' proofreading nucleases in the selective degradation of newly-synthesized DNA and in inducible """"""""error-prone"""""""" replication modes. The behavior of DNA polymerase (lacking the proofreading nuclease) from M. orale on dimer containing phi X174 DNA templates is under investigation. III. We will further test our hypothesis that strand breaks are an obligatory consequence of transcription--e.g., by examining the effect of inducing the lac operon on the accumulation of strand breaks in an F'lac during thymine deprivation. We also are attempting to explain the inviability of polA uvrB mutants and possible anomalous nicking of DNA in these mutants.
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