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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. Memorandum To: Dr. H. McAllister and Staff From: D.H.Ford Re: Site visit with Dr. L. Symington, Columbia University, P & S, July 11,1991. Grant No. 2973 entitled "Molecular and Biochemical Anajysis of DNA Repair." Goals: To determine the molecular and biochemical functions of the RAD50-57 genes which are involved in double-strand break repair, using the yeast Saccharomyces cerevisia as a model system.(RAD = radiation sensitive mutant). Results: Inasmuch as Dr. Symington has only just initiated her CTR supported program, her findings are so far of a developmental and preliminary nature. The studies completed to date are well summarized in the summary progress report in her renewal application and need not be discussed here. Briefly, her efforts were directed toward preparation of an improved method for prep - aring an extract of whole yeast cells which would be competent for in vitro recombinations; developing appropriate substrates for recombination; develop- me h'f'of assays to detect recombination intermediates and their products; and finally the developmenqof yeast strains which contain the pep4 disruption mutation (the pep4 gene encodes one of the vacuolar proteasee). Her new strains of yeast also contain various rad mutations. The background investigations which support Dr. Symington's efforts in her CTR program are covered in a recent publication entitled "Double-strand- break repair and recombination catalyzed by a nuclear extract of S. cerevisae," L. Symington, The EMBO journal 10:987,1991. The summary reports that: An in vitro system for double-strand-break repair and recombination of plasmid substrates catalyzed by extracts of yeast nuclei has been developed. Recombination events which generated crossover products were detected. The recombination . reaction was stimulated by a double-strand break within homologous sequences and preceeded by a mechanism which invo,lved branched DNA intermediates. She noted the presence of pairing events which generated crossovers and the form- ation of inverted repeats (head-to-head and tail-to-tail joined products). In a recent investigation, Dr. Symington has identified a nuclease which clips a single strand piece off of each of two double strands which then fuse together to form a unified combination of the two paired strands, end-to-end. For future consideration is a study wherein she plans to insert mammalian gene sequences into the yeast gene sequence and observe what effect there is on repair of double strand breaks and how it might alter the proteins which are necessary for repair. Comment: Dr. Symington is a young enthusiastic scientist who appears to have a thorough understanding of the overal problem of DNA repair, at least as it applies to yeast, wherein double-strand repair is possible. Inasmuch as the development of malignanecells may be due to failure of DNA repair (following damage caused by some mutagen), the information Dr. Symington obtains with the yeast model may be quite useful in understanding what occurs in higher forms. It will certainly be of interest to note how the presence of mammalian gene sequences influence the synthesis of the yeast proteins involved in repair of strand breaks. An interesting program relevent to the interests of CTR in cancer. DHF