DNA replication cannot take place without active primase. The broad long-term goal of this proposal is to learn enough about primase to develop it as a target to inhibit DNA replication in cancer cells. This proposal continues our studies of the structure, function and mechanism of primer RNA synthesis by E. coli primase and extends our studies to human primase. In the revised proposal, Specific Aim #1 is to study mechanism of movement of E. coli primase on DNA template. A hypothesis of a periodic, punctuated synthesis of pRNA by E. coli primase will be tested by measuring the processivity of primase and dissociation and rebinding of primase to the template after each step of synthesis (10/11 nt, 21/22 nt and 24/25 nt). A primase elongation mutant will be used for these experiments.
Specific Aim #2 is to analyze the active center of pRNA synthesis of E. coli primase. We will use Fe2+ cleavage and site-directed mutagenesis to localize amino acids that bind Mg2+ at the catalytic center and Zn2+ at the zinc finger. The DNA template binding sites will be analyzed using template oligonucleotides containing UV photoreactive nucleotides incorporated in specific positions. The exit pathway of the growing primer RNA chain will be similarly mapped by using photoreactive oligonucleotide primers. Changes in the conformation of primase during pRNA synthesis will be analyzed.
In Specific Aim #3, we will continue to study the role of SSB in pRNA synthesis. A primase/SSB physical interaction will be trapped by biochemical methods. The yeast two-hybrid system will be used to further analyze primase/SSB interaction. Interaction sites on both primase and SSB will be identified.
In Specific Aim #4, we will establish the domain structure of the human primase P49 and 58 subunits of DNA polymerase alpha. The active center of this two- protein system will be identified by ATP affinity cross-linking technique plus chemical cleavage. The function of these regions will be explored by site-directed mutagenesis.
