Somatic hypermutation (SHM) requires the mutagenic potential of the Y-family of DNA polymerases to increase the mutation frequency in immunoglobulin genes. Mistargeted SHM decreases genomic stability and is thought to contribute to B-cell malignancies. However, the absence of function of Y-family polymerases can also result in xeroderma pigmentosum suggesting that properly regulated function is critical for cancer avoidance. The mechanisms targeting Y-family polymerases function are mysterious, but may require protein accessory factors to regulate DNA binding or alter the kinetics of extension from a DNA primer terminus. Using the experimentally tractable Escherichia coli model system, I plan to elucidate the structural and biochemical mechanisms that regulate Y-family polymerase access to or extension from a primer terminus. In E. coli, genetic data suggests that the protein UmuD plays a critical role in regulating DinB, a Y family polymerase member.
Specific Aims :
Aim 1 : Determine the structure of DinB""""""""UmuD2 and DNA""""""""DinB""""""""UmuD2 by X-ray crystallography.
Aim 2 : Study the effect of UmuD and RecA on the DinB reaction pathway for the creation and extension from properly paired and bulged primer termini. Study Design: Using structural and fluorescent methods I will determine mechanisms by which protein regulatory factors alter Y-family polymerase activity. A structure of the DinB""""""""UmuD2 complex bound to DNA will offer key insights into protein""""""""protein and protein""""""""DNA contacts required to activate or prevent DinB-dependent mutagenesis. Furthermore, a detailed structural analysis of the DinB""""""""UmuD2 complex will identify protein conformational changes induced by protein and DNA contacts. Fluorescent pre-steady biochemical studies will complement structural data, allowing a detailed examination of DinB-reaction progression within the context of structural rearrangements induced by protein and DNA contacts. Public Health Relevance Genomic stability requires a specialized set of DNA polymerases that facilitate tolerance of DNA damage until it can be properly repaired. Improper regulation of these polymerases can often result in cancer. This study is designed to elucidate the structural and biochemical mechanisms necessary to regulate one of these specialized DNA polymerases, DinB.
| Foti, James J; Devadoss, Babho; Winkler, Jonathan A et al. (2012) Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics. Science 336:315-9 |
| Foti, James J; Walker, Graham C (2011) Efficient extension of slipped DNA intermediates by DinB is required to escape primer template realignment by DnaQ. J Bacteriol 193:2637-41 |
| Foti, James J; Delucia, Angela M; Joyce, Catherine M et al. (2010) UmuD(2) inhibits a non-covalent step during DinB-mediated template slippage on homopolymeric nucleotide runs. J Biol Chem 285:23086-95 |
