ENZYMATIC MECHANISMS OF DNA SYNTHESIS

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Maturation of mammalian nascent DNA fragments to full length chromosomal DNA is being investigated using purified calf enzymes and synthetic primer-templates. Removal of initiator RNA can be carried out by RNase H1, which specifically cleaves off the intact RNA primer, leaving a monoribonucleotide attached to the DNA segment. This last nucleotide is removed by a 5' to 3' exonuclease, that is stimulated by synthesis from the upstream DNA segment. A nick is generated that is sealed by DNA ligase I. Experiments are proposed to clarify whether the proposed sequence of events is the pathway in vivo. RNA primed DNA made by DNA polymerase alpha/primase, as in vivo, will be tested as a substrate for RNase H1. The above model proposes that these primers, regardless of sequence, be cleaved efficiently and specifically. The ability of the exonuclease to degrade the RNA primer will be evaluated, since that nuclease could substitute for the RNase H. The identity of the DNA polymerase that participates in Okazaki fragment processing is not known. Nuclear polymerases alpha, delta and epsilon will be compared, together with the important co-proteins RF-C, RP-A and PCNA, for ability to work efficiently with the two nucleases and DNA ligase I, to complete the maturation reaction. The 5' to 3' exonuclease also has a unique endonucleolytic capacity. It can cut off an unannealed 5' segment from a primer on a template, only if there is an immediately adjacent upstream primer. This suggests that the initiator RNA could be removed by endonucleolytic cleavage, as the upstream primer is elongated. DNA helicase E promotes displacement from the 5' end of primers, and will be evaluated for ability to promote endonucleolytic primer removal. The exonuclease appears to thread along the unannealed 5' region in order to cleave endonucleolytically. One likely purpose of the endonuclease is to cut past a damaged segment of polymer, however, its motion could be blocked by some types of damage. Tests of its ability to cut past sites modified by cis-platinum and larger adducts, will not only suggest its function in vivo, but will clarify its mechanism of action. The capacity of the endonuclease function to generate substrates for recombination will also be explored. Antibodies will be generated to RNase H1, the 5' to 3' exonuclease and DNA helicase E. These will be used for immunoaffinity purification of these proteins, examination of their cell cycle regulation, and tests of their cellular function by antibody injection into frog oocytes. Homologs of the nucleases and helicase will be identified in S. cerevisiae, where the genes will be disrupted to evaluate cellular role. This thorough approach should define the mechanism of Okazaki fragment maturation.