The main goal of this project to determine the role of protein- protein and protein-DNA interactions in the human base excision- repair (BER) pathway, and to determine how these interactions may vary among polymorphic alleles. BER is the primary cellular mechanism by which single base lesions are repaired. Such lesions result from a variety of endogenous and exogenous agents that cause the oxidation, alkylation, or hydrolysis of bases. For example, A and C residues are spontaneously deaminated by the hydrolysis of the amino groups on the bases; while, oxidative damage by ionizing radiation or treatment with agents such as bleomycin leads to abasic sites and to strand scission, resulting in blocked 3'termini. Both types of damage result in abasic sites in the DNA (the deaminated bases are removed by specific DNA glycosylases) and both are repaired by BER. BER in humans is initiated by AP endonuclease which hydrolyticly cleaves the phosphodiester bond 5' to the abasic site, leaving a 5' dRP site and a 3'OH. DNA polymerase beta subsequently extends the DNA chain by one nucleotide and removes the dRP site with its dRP lyase activity, resulting in a double-stranded DNA containing a nick. The nick is subsequently sealed by a DNA ligase (either DNA ligase I or DNA ligase III in the presence of the repair protein XRCC1). BER involves a complex set of interactions between the repair proteins and DNA. AP endonuclease, DNA pol beta, and DNA ligase I (or III) all catalyze reactions on DNA. In addition, in vitro evidence indicates that DNA pol beta binds to DNA ligase I and to xrcc1, which binds to DNA ligase III. Finally, AP endonuclease and xrrc1 have been shown to affect the activity of DNA pol beta. These observations suggest a process wherein both a spatial and temporal sequence of binding events govern the efficiency of BER concerted DNA repair. To understand how genetic polymorphisms in BER genes might affect DNA repair, it is paramount to understand the details of their interactions. To further elucidate the mechanisms involved in the macromolecular interplay of BER, we are prepared to invest considerable effort towards the biophysical solution-state analyses, as well as scanning force microscopy analyses of the components of the BER system. Such knowledge at this molecular level will obviously impart directly a much greater understanding at the cellular level and a greater perspective of the importance of BER concerted DNA repair. The specific questions that we will address are: 1) How are the conformations of the protein-DNA complexes affected by the presence of the other proteins involved in the repair? 2) What are the binding affinities of the proteins for one another and are they affected by the presence of damaged DNA. 3) How do polymorphisms affect the above properties?
| Yang, Yong; Wang, Hong; Erie, Dorothy A (2003) Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy. Methods 29:175-87 |
| Xue, Yu; Ratcliff, Glenn C; Wang, Hong et al. (2002) A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3'- 5' exonuclease and 5'-protruding strand endonuclease activities. Biochemistry 41:2901-12 |
| Hall, M C; Wang, H; Erie, D A et al. (2001) High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer. J Mol Biol 312:637-47 |
| Ratcliff, G C; Erie, D A (2001) A novel single-molecule study to determine protein--protein association constants. J Am Chem Soc 123:5632-5 |
