The goals of this project are to understand recombinational DNA repair, particularly the mechanismsof the central recombinases involved in this process and their regulation. Homologous geneticrecombination is at once (a) a key DNA repair process, (b) one of the important cancer avoidancepathways in higher eukaryotes, and (c) one of the primary paths to the productivealteration/engineering of cellular genomes. This competing proposal is focused on the bacterial RecAprotein, proteins that regulate or augment RecA function, and proteins that function in closely relatedpathways. We will also explore specific applications of these proteins in biotechnology. There are four specific aims. Aim 1 is focused on RecA itself. We will generate RecA variantswith enhanced functionality, which may eventually find application in biotechnology. Aims 2 and 3 focus on proteins that have a major role in maintaining genome stability, butaddress molecular functions that have been largely overlooked. The subject of Aim2 is the proteinMgsA (maintenance of genome stability A). MgsA has close homologues in all organisms frombacteria to humans, but its function has been enigmatic. We have found that it opens up DNA ends,presumably to facilitate the productive loading of key helicases that function in DNA replication. Aim3 addresses a previously uncharacterized protein called RadD. This protein may play a role inremoving barriers such as RNA polymerase from damaged DNA, clearing the way for DNA repairprocesses. Finally, Aim 4 will continue our efforts to elucidate the role of RecA protein in the function ofthe mutagenic DNA polymerase V. We have made much progress in the past 4 years on ourunderstanding of this unusual DNA polymerase, and are now down to mapping the molecularinteraction between RecA and the polymerase subunits. The RecA variants generated in Aim 1 may have some practical application as the subjects ofnew studies to explore RecA structure/function and as reagents in RecA applications in forensicscience and genome engineering.