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

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DNA damage-induced mutagenesis in E. coli occurs transiently as part of the global SOS response. A key participant in this process is the UmuD' protein which, together with UmuC and activated RecA (RecA*), enables the DNA polymerase III holoenzyme to replicate across chemical and UV-induced lesions. Our x-ray crystallographic analysis shows that in addition to forming molecular dimers, the amino-terminal and carboxyl-terminal tails of UmuD' extend from a globular beta structure to associate and produce crystallized filaments. These filaments are required for biological activity: Deletion of the amino terminus does not affect the ability of the protein to form molecular dimers but does severely compromise the ability of UmuD' to interact with a RecA-DNA filament and to participate in mutagenesis. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized S. cerevisiae DNA repair gene related to the E. coli umuC, dinB, and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Our findings suggest that RAD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. We previously identified umu-complementing genes on two incL/M plasmids, R471a and R446b. Molecular analysis of these genes reveals that they are more structurally and functionally related to mucAB than to other members of the umu-like family. We found an insert 5' to mucAB(R471a) that appears to be a novel retroelement encoding a putative reverse transcriptase (RT); this RT is related to the RTs encoded by group II introns but it is embedded in a retron-like context. This observation suggests that the mucAB-like gene in R471a is located within a region of the R-plasmid that perhaps was once (or still is) a mobile genetic element. This observation might explain the distribution of umu-like genes on R-plasmids and bacterial chromosomes. In work on the repair of UV-induced DNA damage in primate cells, we have focused on UV-DDB, a protein complex comprising p127 and p48 subunits that binds specifically to 6-4 pyrimidine dimers (PD). This binding activity is not detectable in xeroderma pigmentosum (XP) Group E; repair can be restored in XP-E cells by microinjection of UV-DDB. However, in contrast to all other XP proteins, UV-DDB does not promote repair in an in vitro reconstituted repair system (naked DNA substrate). We have now found that another member of the UV-DDB complex is RPA, a known damage-recognition protein. Since UV-DDB is required for in vivo but not in vitro DNA repair, we hypothesize that UV-DDB may recognize and bind to 6-4 PDs and alter the surrounding chromatin structure to permit access of the "repairosome".