Homologous recombination is generally considered a major mechanism by which cells repair many types of DNA lesions and damaged replication forks. However, if this process is left unchecked, cells often show a hyper-recombination (hyper-rec) phenotype, and are susceptible to large deletions, duplications, or inversions of important genetic information.

This dissertation describes two projects aimed at examining molecular mechanisms by which cells regulate homologous recombination. The first shows several 3'-5' exonucleases prevent RecA-GFP loading by destroying potential substrates. It is shown that two genetic pathways exist: one consisting of ExoIII and another comprised of ExoVII, ExoIX, ExoX, and ExoXI. ExoI acts upstream of both of these pathways. Although xthA cells have an increase in DSBs and recB-dependent loading of RecA-GFP, they are viable with a recB mutation and do not display a large increase in SOS expression. The increase in RecA-GFP is also independent of base excision repair (BER). These experiments uncovered that DNA in a population of wild type cells undergoes DSBs and is often repaired in a RecA-independent manner after processing by ExoI and ExoIII.

The second project shows the helicase, UvrD limits the number and intensities of RecA-GFP foci. This activity is due to the ability of UvrD to remove RecA from DNA where it is loaded in a RecF pathway-dependent manner. This activity requires ATP binding by UvrD, suggesting that helicase/translocase activity is important for RecA-removal. The hyper-helicase mutation, uvrD303 confers UV sensitivity to cells. Epistasis analyses showed uvrD303 is defective in the recA pathway of UV repair and not in nucleotide excision repair (NER). Surprisingly, UvrD303 does not directly remove RecA after UV, as new RecA-GFP foci appear like in wild type cells. UvrD303 does, however, slightly inhibit SOS induction, and constitutively activating the SOS response restores UV resistance to these cells in a way that is independent of recA overexpression. Furthermore, uvrD303 was capable of suppressing the constitutive SOS phenotype of recA730. These experiments suggested that UvrD303 antagonizes the ability of RecA filaments to induce the SOS response, rendering cells UV sensitive.