DNA REPLICATION, REPAIR, AND MUTAGENESIS IN EUKARYOTIC AND PROKARYOTIC CELLS

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Studies on the mechanism of mutagenesis in prokaryotic cells focused on RecA and UmuDC-like mutagenesis proteins. We crystallized the UmuD' protein; refined the structure to 2.5 angstroms; and elucidated the mechanism of self-cleavage which converts UmuD to the mutagenically active UmuD'. We also discovered that while UmuD' forms a molecular homodimer, it can also form an extended polymeric structure which interacts with RecA* as well as UmuC, facilitating translesion DNA synthesis at the site of unrepaired DNA damage. We found that UmuD and UmuC proteins are inherently unstable in E. coli and are degraded by the Lon protease. UmuC is, however, protected when co-expressed with UmuD'. While the homodimeric UmuD' protein is quite stable, it is rapidly degraded by the ClpXP protease when in a heterodimeric complex with UmuD. These interactions allow error-prone, Umu-mediated synthesis only as a last resort, avoiding gratuitous mutagenesis. Studies with X.laevis demonstrated that while oocytes can efficiently replicate undamaged ss DNA, they are unable to replicate DNA which contains adducts. This arrest was alleviated in progesterone-matured oocytes and in oocytes microinjected with umuDC mRNAs, suggesting that the basic mechanisms of mutagenesis are highly conserved. We analyzed the specificity of the mutator phenotype of a recA730 lexA51(Def) strain by employing F_-plasmids carrying a set of mutant lacZ genes that can individually detect specific transitions, transversions, and frameshift events. Most of the spontaneous mutagenesis could be attributed to a specific increase in A:T to T:A, A:T to C:G and G:C to T:A transversions. These events were completely abolished in a delta umuDC strain, indicating that functionally active UmuD'C proteins are normally required for their generation. The spectrum obtained was similar to that of strains with a defect in the epsilon (3'-5' proofreading) subunit of DNA polymerase III, raising the possibility that the wild-type epsilon protein is inactivated in strains expressing the RecA730 and UmuD'C proteins. We also used an ss DNA vector carrying a site-specific T-T cyclobutane dimer to analyze the mutagenic specificity of a set of isogenic E. coli delta umuDC strains harboring low-copy-number plasmids expressing UmuD'C or its homologs (mucA'B, rumA'B). In umuD'C strains, 3' T to A mutations outnumbered 3' T to C, but the reverse was true for the homologs, suggesting that UmuD'C and its homologs may differ in their relative abilities to promote elongation from mismatched termini. To date, eight closely related homologs of the E. coli UmuC protein have been identified, with all appearing to play critical roles in damage-inducible mutagenesis in enterobacteriaceae. A distantly related UmuC-homolog, DinB, was also identified in E. coli, as was a new member of the UmuC-superfamily in the archeon, Sulfolobus solfataricus [dbh (dinB homolog)]. In a second project, we studied a protein complex (UV-DDB/XPE factor) which binds to 6/4 photoproducts in UV-damaged human DNA. We found, in an in vitro nucleotide excision repair (NER) assay, that the addition of the complex leads to a modest stimulation of repair, suggesting that UV-DDB plays an accessory, but not a core role, in the NER process. We also found that the complex moves to a tight association with damaged DNA upon UV treatment of cells. RPA, which also redistributes after UV, is also present in the UV-DDB/DNA complex, and the interaction of DDB and RPA enhances the DNA binding of either alone, suggesting that UV-DDB functions in the initial recognition step of DNA repair.