The long term goal of this proposal is to establish a detailed in vitro replication system for chloroplast (ct) DNA. Replication of ctDNA proceeds by the D-loop mechanism and involves both Cairn's replicative intermediates and those replicative intermediates produced by the rollig circle mechanism. A replication transcription complex (RTC) from chloroplasts that consists of 30-50 polypeptides has been isolated. This complex can be obtained either with endogenous ctDNA (DNA-RTC comlex) or without the DNA (RTC complex). A DNA polymerase, a DNA binding protein, and an RNA polymerase were purified from this complex. The first phase of research will concentrate on initiation of ctDNA replication and elongation of the newly synthesized DNA chains. Sites of initiation of replication will be identified on the restriction endonuclease map of pea ctDNA by cross linking the supercoiled DNA with trioxsalene, followed by restriction and electron microscopic analysis of the DNA fragments containing D-loops. Precise position of D-loops will be determined by in vitro 5 feet end labeling using polynucleotide kinase and Gamma-labeled dTTP, purifying the labeled D-loops in denaturing gels, hybridizing the D-loops with an appropriate restriction fragment of ctDNA, single strand nuclease digestion, and analysis in the DNA sequencing ladder. The in vitro replication process will be studies using RTC comlex and supercoiled DNA as a template. IN parallel with these studies, we will purify two more enzymes from the RTC complex. The first enzyme will be topoisomerase, whose presence in chloroplasts is already identified. The second enzyme will be DNA ligase. Using topoisomerase, DNA polymerase, DNA binding protein, and DNA ligase, we will attempt to reconstitute the system to see whether DNA synthesis takes place using supercoiled ctDNA. It is hoped that such a system will at least carry out extension of already formed D-loops because D-looped molecules resemble a gapped DNA molecule, which is the preferred substrate of the ctDNA polymerase.
| Nielsen, B L; Lu, Z; Tewari, K K (1993) Characterization of the pea chloroplast DNA OriA region. Plasmid 30:197-211 |
| Nielsen, B L; Rajasekhar, V K; Tewari, K K (1991) Pea chloroplast DNA primase: characterization and role in initiation of replication. Plant Mol Biol 16:1019-34 |
| Meeker, R; Nielsen, B; Tewari, K K (1988) Localization of replication origins in pea chloroplast DNA. Mol Cell Biol 8:1216-23 |
| Gold, B; Carrillo, N; Tewari, K K et al. (1987) Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis. Proc Natl Acad Sci U S A 84:194-8 |
| Tewari, K K (1986) Purification and properties of chloroplast DNA polymerase. Methods Enzymol 118:186-201 |
