This project encompasses a number of separate research problems relevant to our understanding of molecular mechanisms for replication and processing of DNA. Research effort currently includes examination of the following problems: (a) Replication of the terminal segment in the E. coli chromosome. Our studies on the effect of temperature shifts on the incorporation of labeled thymidine into DNA in E. coli do not support the hypothesis that a unique terminal segment exists and that protein synthesis is required for the synthesis of that segment. We are now examining the effect of temperature shifts on rates of DNA replication. The size and specific activity of the thymidine triphosphate pool is being studied using a two-dimensional thin layer chromatography system. Attempts are also being made to develop a one-dimensional system suitable for use with 32P-labeled cell extracts. (b) The mechanism of action of nalidixic acid is being studied in E. coli, particularly in relation to its effect on the completion and joining of Okazaki fragments. (c) Thymineless death and DNA "turnover" in bacteria. We are testing our hypothesis that thymineless death (tld) results from the accumulation of unrepaired single-strand breaks in DNA. Similarities between tld and the lethality of dnaF strains (defective in ribonucleoside reductase) are being examined. Our hypothesis that transcription introduces repairable single strand breaks in bacterial DNA is being tested by selective inhibition of transcription in RNA polymerase deficient mutants or by rifamprin, puromycin, or chloramphenicol treatment and subsequent examination of DNA molecular weight distribution by alkaline sucrose gradient sedimentation. (d) Intermediates in replication in SV40 DNA are being analyzed following ultraviolet irradiation of the virus-host cell complex using the T4-endonuclease V to monitor the distribution of pyrimidine dimers in parental and daughter DNA. BIBLIOGRAPHIC REFERENCES: Sarasin, A. and P. Hanawalt "DNA repair in human cells treated with activated aflatoxin B1" Mutation Res. (in press) (1977). Little, J. and P. Hanawalt "Induction of protein X in E. coli" Mol. Gen. Genetics (in press) (1977).