The purpose of this proposal is to study the mechanism of replication of the chromosome, especially in relation to the possible involvement of RNA primers iun discontinuous DNA synthesis. A new method has been developed for the selective isolation of newly synthesized DNA from permeable cells. This involves the use of 50mercuri-dCTP as substrate for the isolation of nascent DNA chains by affinity chromatography on thiol-agarose. Using this new methodology, DNA replication in Bacillus subtilis cells permeabilized with toluene will be investigated. Newly synthesized DNA will be isolated and analyzed for the presence of 5'-terminal RNA primer. Biochemcial characterization of the primers will include the determination of overall base composition, chain length and nucleotide sequence. Other important questions to be answered concern the fraction of newly synthesized DNA chains which are linked to RNA primer and the kinetics of RNA primer synthesis and degradation, using various mutants. Besides various properties of mercurated DNA will be examined, such as biological activity during transformation, cleavage by restriction endonucleases and electron microscopy. A second major application of the new methodology will be the study of chromosomal replication in mammalian cells. Mouse L cells permeabilized with dextran sulfate will be used to characterize DNA synthesis with HgdCTP to ascertain whether it represents chromosome replication. We will then attempt to isolate and characterize possible RNA primers involved in discontinuous DNA synthesis. The nature of the initiation sequences of the nascent DNA fragments will also be determined and compared with the various types of DNA sequences found in the chromosome. These studies will be extended to 3T6 and CV-1 cell lines which are virus hosts. The proposed studies using a novel approach should provide important information about replication intermediates in prokaryotes and eukaryotes.
