The long range goal of this proposal is to understand the molecular mechanism of DNA replication and the processes which regulate DNA synthesis in the eukaryotic cell. This knowledge is fundamental to our understanding of such important questions as the mechanism of viral replication and the growth of both normal and malignant cells. As a model system, the enzymes which function in DNA replication in the yeast Saccharomyces cerevisiae will be studied. DNA polymerase I has been purified about 10,000 fold from a protease deficient strain of yeast. A DNA primase activity, which synthesizes short RNA primers for initiation of DNA synthesis on single-stranded DNA templates, copurifies with the DNA polymerase. This study will focus on these two enzymatic activities and on additional protein factors which may enhance DNA synthesis on primed and unprimed single-stranded DNA templates. The subunit composition of the polymerase-primase will be investigated by raising antibodies to the major polypeptides present in DNA polymerase I preparations and by recovering enzymatic activity from SDS polyacrylamide gels. These and other studies will determine whether the polymerase and primase activities are contained in a single polypeptide or in separate subunits of the enzyme. In preliminary experiments, a primase activity has also been observed in fractions containing no DNA polymerase activity. This primase will be purified and its physical and enzymatic properties will be compared with the properties of the polymerase-primase complex. A search will be made for protein factors which affect the efficiency and/or specificity of the primase reaction and for factors which increase the processivity of the polymerase. Such factors may be found associated with the DNA polymerase in a complex analagous to the E. coli DNA polymerase III holoenzyme. Finally, the gene(s) encoding DNA polymerase I, and possibly other replication proteins, will be isolated using antibodies raised against the purified protein to screen a yeast DNA library cloned in the expression vector Lambda gt11. The cloned gene will be sequenced and used to generate mutants to establish a role for the enzyme in DNA replication.
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| Lawrence, C W; Hinkle, D C (1996) DNA polymerase zeta and the control of DNA damage induced mutagenesis in eukaryotes. Cancer Surv 28:21-31 |
| Singhal, R K; Hinkle, D C; Lawrence, C W (1992) The REV3 gene of Saccharomyces cerevisiae is transcriptionally regulated more like a repair gene than one encoding a DNA polymerase. Mol Gen Genet 236:17-24 |
| Brooke, R G; Singhal, R; Hinkle, D C et al. (1991) Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities. J Biol Chem 266:3005-15 |
