of Work: Initiation of human DNA replication entails a highly regulated assembly of polymerase complexes along with chaperone proteins and other enzymes. The polymerase alpha with its associated primase activity is the only polymerase involved in this initiation process. This enzyme complex is composed of four proteins, a 180 kDa phospho-glyco protein containing the DNA polymerase activity, a 70 kDa phosphoprotein with no known function, and two smaller subunits of 49 and 58 kDa containing the primase activity. Current research is focused on understanding the role of conserved amino acids in the active site of the catalytic polymerase subunit in the fidelity of DNA replication. Alignment of characteristic family A and B polymerases over motif A suggested that Y865 of human pol a may function as E710 in E. coli DNA pol I which is critical for ribose discrimination. The effect of this change, Y865S, has been analyzed for the incorporation of ribonucleotides. Y865 has been changed to alanine and two additional mutant proteins, Y957A and Y957F, were produced. Tyrosine-957 is functionally analogous to Tyr766 in E. coli DNA pol I which is important for fidelity. These mutant proteins have been overproduced in our baculovirus system, purified and characterized for their polymerase function and DNA synthetic fidelity.
| Burgers, P M; Koonin, E V; Bruford, E et al. (2001) Eukaryotic DNA polymerases: proposal for a revised nomenclature. J Biol Chem 276:43487-90 |
