The long term goal of this project is to gain a better understanding of the mechanism of DNA replication by reconstruction in vitro of events that take place at the replication fork. Our recent progress toward this goal has included: preparation of intact, homogeneous DNA polymerase alpha/primase; purification to homogeneity of a new accessory factor for DNA polymerase alpha/primase (alpha accessory factor or AAF) and characterization of its properties and novel mechanism of action; development of a system that emulates discontinuous DNA synthesis on the lagging strand using five purified mouse proteins; identification of a RNA primer removal mechanism; and purification and characterization of two DNA polymerases delta from mouse cells, one of which is the catalytic subunit of the other. It is proposed that this work be continued as follows: 1) Carry out additional studies on several of the proteins thus far purified; particularly, AAF, the 5'-exonuclease, DNA polymerase alpha/primase, and RNase H-1. This would include scale-up of the purification procedure to make available larger amounts of protein; more detailed studies on physical and catalytic properties and mechanism of action; preparation of antibodies as a reagent for polypeptide identification and specific inhibition of activity; and selection of cDNA as a source (through overexpression) of the protein and as another route to information on structure and function. A special effort will be made to clarify the roles of putative replication proteins, e.g. AAF, the 5'- exonuclease and RNase H-1. 2) Purify and characterize additional replication proteins by specific assays; this includes helicases and additional RNA primer-removing activities. 3) Develop an in vitro DNA replication system dependent on Epstein-Barr virus (EBV) oriP and EBNA-1, to study its components and mechanism. 4) Construct a preformed replication fork on which to synthesize leading and lagging strands from nine purified proteins; this, as well as the EBV system, will be used to detect, purify and characterize additional proteins that participate in DNA replication. It is believed that greater knowledge of the mechanism of DNA replication will result in health benefits because of its central importance in problems such as errors in replication and injury/repair of DNA and their relationship to mutations and carcinogenesis, as well as providing a basis for the rational design and use of chemotherapeutic agents. In particular, it is hoped that an understanding of the structure and mechanism of EBV oriP and EBNA-1 and the cellular proteins with which they interact will contribute to knowledge concerning the pathogenesis of EBV-related tumors and ultimately help in their control.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA011705-22
Application #
3163544
Study Section
Biochemistry Study Section (BIO)
Project Start
1991-04-01
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
22
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Goulian, M; Heard, C J (1991) Discrimination between mammalian RNases H-1 and H-2. Anal Biochem 192:398-402
Goulian, M; Heard, C J (1990) The mechanism of action of an accessory protein for DNA polymerase alpha/primase. J Biol Chem 265:13231-9
Goulian, M; Heard, C J (1990) An inhibitor of DNA polymerases alpha and delta in calf thymus DNA. Nucleic Acids Res 18:4791-6
Goulian, M; Heard, C J; Grimm, S L (1990) Purification and properties of an accessory protein for DNA polymerase alpha/primase. J Biol Chem 265:13221-30
Goulian, M; Richards, S H; Heard, C J et al. (1990) Discontinuous DNA synthesis by purified mammalian proteins. J Biol Chem 265:18461-71
Goulian, M; Herrmann, S M; Sackett, J W et al. (1990) Two forms of DNA polymerase delta from mouse cells. Purification and properties. J Biol Chem 265:16402-11
Goulian, M; Grimm, S L (1990) Three cytoplasmic DNA polymerases that utilize poly(rA).oligo(dT). Biochem Biophys Res Commun 170:627-34
Goulian, M; Heard, C J (1989) Intact DNA polymerase alpha/primase from mouse cells. Purification and structure. J Biol Chem 264:19407-15
Goulian, M; Bleile, B M; Dickey, L M et al. (1986) Mechanism of thymineless death. Adv Exp Med Biol 195 Pt B:89-95
Ingraham, H A; Dickey, L; Goulian, M (1986) DNA fragmentation and cytotoxicity from increased cellular deoxyuridylate. Biochemistry 25:3225-30