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. Our research is concentrated on understanding the regulation and mechanism of the human DNA polymerase alpha - primase complex. 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 the enzymatic mechanism and regulation of the two primase subunits. Previously we had cloned and overexpressed the cDNA for the two human DNA primase subunits (Hp49 and Hp58) and purified the proteins from E. coli. This overexpression system was used to study the protein interaction between the two primase subunits. We determined that protein-protein contacts between the two subunits are made over several domains within the Hp58 subunit and most of this interaction is essential for primer initiation by the Hp49 subunit. With our development of a high yield expression system we have entered into a collaboration with Dr. Silvia Onesti (Imperial College, London) to crystallize the two human primase subunits and determine the three dimensional structure. Using highly purified primase subunits in single form and as a heterodimeric complex, crystal growth conditions are being discovered for optimal X-ray diffraction. The three dimensional structure of the primase complex will help elucidate the complex chemistry of primer formation and open up the field for structure specific design of inhibitors which possibly may be used in chemotheropeutics. We have also entered into a collaboration with Dr. Rob Kuchta, Bolder, CO, using Hp58 deletion mutants to study the role of Hp58 in primer initiation.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Intramural Research (Z01)
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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