Title

Studies of the Chloroplast DNA Polymerase: Association of Exonucleases, Processivity, Fidelity, and Identification of the Catalytically Active Polypeptide

Date of Award

1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Gordon C. Cannon

Advisor Department

Chemistry and Biochemistry

Abstract

Chloroplast DNA (ctDNA) replication, an essential process for the survival of photosynthetic organisms, is poorly understood at the molecular level, partially because of the lack of knowledge concerning the central enzyme in this process: the ctDNA polymerase. This study attempted to provide a better understanding of ctDNA replication through two different, yet parallel, experimental approaches involving the ctDNA polymerase. The first approach was biochemical analysis of a genetically characterized Arabidopsis thaliana mutant, chm1, thought to be the product of a defect in the ctDNA polymerase gene. The second approach involved biochemical characterization of the two forms of soybean ctDNA polymerase with respect to association with a 3$\sp\prime$ to 5$\sp\prime$ exonuclease, processivity, and fidelity. The Arabidopsis thaliana chloroplast mutator (chm1), characterized by George Redei as a nuclear point mutation demonstrating recessive Mendelian inheritance, is identified by the production of a "yellow, white, green variegated phenotype" (1). The various mutant phenotypes are stabilized by back-crossing to plants with a wild-type nuclear background. The stabilized mutant phenotypes exhibit maternal cytoplasmic inheritance. The nuclear point mutation, thought to be in the ctDNA polymerase gene, is hypothesized to reduce the fidelity of the ctDNA polymerase. Comparison of partially purified ctDNA polymerase activity isolated from 10-day-old chloroplasts of homozygous chm1 and wild-type Arabidopsis thaliana plants demonstrated similar levels of nucleotide misincorporation when assessed by a gel-based assay. This suggested that the nuclear point mutation does not alter the fidelity of the ctDNA polymerase and probably does not reside in the ctDNA polymerase gene. Biochemical characterization of the two forms of ctDNA polymerase partially purified from isolated chloroplasts of soybean from the SB-1 cell suspension culture exhibited differences in association with 3$\sp\prime$ to 5$\sp\prime$ exonuclease activity, processivity, and fidelity Form I ctDNA polymerase copurified with a dissociable 3$\sp\prime$ to 5$\sp\prime$ exonuclease activity, incorporated 80 nucleotides per association event, and synthesized DNA with a lower fidelity than Form II polymerase which lacked the 3$\sp\prime$ to 5$\sp\prime$ exonuclease and incorporated 262 nucleotides per association event. Both forms of ctDNA polymerase lacked a 5$\sp\prime$ to 3$\sp\prime$ exonuclease, suggesting neither form was capable of performing nick translation. Both forms of soybean ctDNA polymerase were found to associate with chloroplast nucleoids. Taken together, these data strengthen the argument that the two forms represent different in vivo states of the ctDNA polymerase and probably are involved in ctDNA replication.