BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF THREE DNA REPAIR ENZYMES IN DEINOCOCCUS RADIODURANS
Julin, Douglas A
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The Gram positive bacterium Deinococcus radiodurans is able to withstand acute doses of gamma rays that can cause hundreds of double-strand breaks per genome. In proposed double-stand break repair pathways, however, some important enzymes, such as helicases and nucleases in the initiation step, have not been clearly identified yet. Interestingly, the common bacterial helicase/nuclease complex RecBCD or AddAB, which functions to produce a 3' ssDNA tail in double-strand break repair initiation step in other bacteria, is not found in D. radiodurans. As part of efforts to identify helicases involved in double-strand break repair, the D. radiodurans HelIV (encoded by locus DR1572, the helD gene) was characterized with both in vivo and in vitro methods. The helD gene is predicted to encode a helicase superfamily I protein. The helD mutant is moderately sensitive to methyl methanesulfonate and hydrogen peroxide but it is not sensitive to gamma rays, UV and mitomycin C. In biochemical assays, the full length HelIV exhibited DNA unwinding activity with a 5'-3' polarity whereas the truncated HelIV without N-terminal region had no detectable helicase activity. RecJ is the exonuclease in the RecF pathway, which is suggested to function at the initiation step in DSB repair in the absence of RecBCD. In the in vivo study, the D. radiodurans recJ gene (encoded by locus DR1126) cannot be completely removed from the chromosome, indicating the essential role of RecJ in cell growth. The heterozygous mutant displayed growth defect and higher sensitivity to gamma rays, hydrogen peroxide and UV compared to wild type D. radiodurans, suggesting an important role in DNA repair. The RecJ expressed in E. coli system was insoluble but can be purified via denaturation-refolding, and the refolded RecJ showed 5'-3' exonuclease activity. D. radiodurans has no RecB and RecC proteins, but it has a homologue of the RecD protein. We tested whether the D. radiodurans RecD protein could form a complex or make transient interactions with other proteins to perform more complicated functions. The RecD conjugated protein affinity column was used to attempt to identify cellular binding partners.