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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025022-08
Application #
3272723
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1978-09-01
Project End
1988-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
8
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472
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Taljanidisz, J; Popowski, J; Sarkar, N (1989) Temporal order of gene replication in Chinese hamster ovary cells. Mol Cell Biol 9:2881-9
Basnakian, A; Banfalvi, G; Sarkar, N (1989) Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase. Nucleic Acids Res 17:4757-67
Taljanidisz, J; Decker, R S; Guo, Z S et al. (1987) Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains. Nucleic Acids Res 15:7877-88
Sarkar, N; List, J F; Banfalvi, G (1987) Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells. Eur J Biochem 168:263-8
Banfalvi, G; Sarkar, N (1985) Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis. J Mol Biol 186:275-82
Banfalvi, G; Bhattacharya, S; Sarkar, N (1985) Selective isolation of mercurated DNA by affinity chromatography on thiol matrices. Anal Biochem 146:64-70