This project seeks to understand the molecular mechanism of non-template-directed nucleotide addition by human DNA polymerases at DNA termini. It therefore seeks to understand the basic problem of errors in nucleotide selection by DNA polymerases from a novel perspective. Non-template-directed nucleotide addition is the ability of some DNA polymerases to synthesize DNA in spite of the absence of a template to direct the preferential insertion of a particular nucleotide. This can occur at DNA ends, especially ends created by either damaging agents (e.g. ionizing radiation) or replication fork collapse. How DNA polymerases interact with strand breaks and synthesize DNA in human cells is not fully known. To address this problem we have designed a set of experiments to test whether human DNA polymerases from three different structural families of polymerases can perform non-templated addition. The projects described here use an in vitro approach to examine what structural features of DNA ends are critical for non-templated addition in each polymerase. Also, experiments are proposed to examine whether other proteins, important in replication and repair, are necessary for non-templated addition. The goal is to have a more complete understanding of how nuceleotides are added to growing DNA molecules by DNA polymerases. The significance of understanding how human DNA polymerases add nucleotides without template direction rests in the insights it will shed on our understanding of molecular basis of mutagenesis and biological processes like cancer and aging. As a comparative model, it will also improve our understanding of nucleotide normal DNA synthesis works. This proposal has a strong educational component in that is seeks to encourage undergraduate participation, especially from those groups who have been traditionally under- represented in academic research. ? ? ?
