The central objective of this project is the analysis of error-prone repair in bacteriophage T4, with related explorations into other mechanisms of mutagensis and into a novel mechanism for avoiding ultraviolet-induced killing. Most chemical and all radiation mutagenesis in T4 occurs via error-prone repair and depends on the functions of the genes uvsW, uvsX and uvsY, most or all of the genes required for DNA replication, and perhaps several other genes as well. We have recently characterized temperature-sensitive alleles of the X and Y genes, alleles that differentially affect mutagenesis and inactivation. We are now cloning these mutant genes in order to obtain their proteins in amounts sufficient to determine which kinetic parameters correlate with mutagenesis and which with survival. Temperature-sensitive alleles of uvsW have also been obtained and will be characterized as to survival and mutation after UV irradiation; at the same time, a clone of the uvsW gene is being trimmed down to minimum size for further studies of the structure and function of this gene. We plan to explore which other genes may be required for error-prone repair in T4, with particular attention to genes 46, 47, 49, 58 and 59, and to continue with mapping experiments to localize a mutation, hm, which promotes ultraviolet mutagenesis. Experiments will be conducted to determine whether error-prone repair and genetic recombination are correlated, both processes being controlled by genes of the WXY pathway. Finally, our work on a recently discovered new mode of survival after UV irradiation will be prepared for publication; this system involves the genes encoding the ssDNA-binding and helicase proteins.
