The long-term goal of this application is to understand biochemical mechanisms operating at a DNA replication point that contribute to mutagenesis in the bacterium, Salmonella typhimurium. Classical studies on this subject have focused on three areas: base misinsertion on normal templates, base misinsertion on damaged templates, and editing of misinserted bases. Few studies have dealt with mechanisms allowing continuation of synthesis once a base has been misinserted, even though available evidence indicates that a mismatched primer terminus should form a significant barrier to extension. It is generally thought that when many mismatched primer termini must be extended, successful replication depends on a transient alteration in some property of the polymerase. The specific nature of such an alteration is unknown. In this application, attention will focus on the in vitro analysis of a stable genetic varient of DNA polymerase III, altered in its alpha (polymerization) subunit, that is able to efficiently replicate DNA in vivo despite a complete lack of editing capacity (resulting from a deletion of the gene for the editing (epsilon) subunit).
The specific aims are as follows. First, the pol III alpha subunits from wild-type and variant cells will be compared in detail with respect to several enzymatic properties. These studies will provide a picture of the specific nature of the lethal defect caused by the absence of epsilon protein and suppressed by the mutation in the gene for alpha. Second, since the alpha variation suppresses the lethality of the epsilon deletion, the ability of epsilon to modulate wild type alpha activity to make it more like the variant alpha will be examined. The third specific aim is to define structural correlates in epsilon for the exonuclease and alpha-modulation functions by examining the properties of epsilon fragments.
The fourth aim i s to relate the properties of the variant alpha to the process of SOS mutagenesis, in which the proposed modification of polymerase activity is determined by the umuCD genes. The relationship will be explored by determining whether the variant alpha obviates the requirement for umuC and/or umuD for UV mutagenesis and Weigle reactivation.
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|Lifsics, M R; Lancy Jr, E D; Maurer, R (1992) DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III. J Bacteriol 174:6965-73|
|Slater, S C; Maurer, R (1991) Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium. Proc Natl Acad Sci U S A 88:1251-5|
|Lancy, E D; Lifsics, M R; Kehres, D G et al. (1989) Isolation and characterization of mutants with deletions in dnaQ, the gene for the editing subunit of DNA polymerase III in Salmonella typhimurium. J Bacteriol 171:5572-80|
|Lancy, E D; Lifsics, M R; Munson, P et al. (1989) Nucleotide sequences of dnaE, the gene for the polymerase subunit of DNA polymerase III in Salmonella typhimurium, and a variant that facilitates growth in the absence of another polymerase subunit. J Bacteriol 171:5581-6|