Examining the Role of Exo V in Repairing DNA Damage

University Of Miami School Of Medicine, Coral Gables FL

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Abstract

DESCRIPTION (provided by applicant): Life depends upon the faithful transmission of genetic information from one generation to the next. Unfortunately DNA damage occurs and, when it does, it can lead to the loss of important genetic information. My work focuses on understanding how two different groups of bacteria have chosen to solve the problem presented by one very harmful type of DNA damage: double-strand breaks. The first part of this proposal focuses on RecBCD, which is a heterotrimeric protein from E. coli. RecBCD is a potent double strand 5'-3' and 3'-5' nuclease, helicase, and ATPase. It is highly processive, digesting kilobases of DNA per binding event. It is, however, also a recombinase that promotes homologous recombination. These two paradoxical roles are mediated by the 8-nucleotide chi sequence (5'-GCTGGTGG-3'). The second part of the proposal focuses on AddAB, the two-subunit enzyme responsible for Exo V activity in T. tengcongensis and other gram-positive bacteria. AddAB has the same activity and role in the cell as RecBCD except it responds to a different chi sequence. In this project I will use a variety of techniques including crystallography, genetics, and enzymology to determine the structure and dissect the regulation of these processive nucleases.