Isolation and characterization of an in vitro DNA replication system from maize mitochondria.
An in vitro DNA replication system from maize mitochondria has been isolated and characterized. Maize mtDNA polymerase activity was purified about 1100-fold through DEAE cellulose and Heparin-Sepharose columns. In addition to the DNA polymerase activity, this in vitro replication system also contained topoisomerase I, DNA primase and RNA polymerase activities. Optimal conditions for enzyme activity, preferred templates and inhibitors were determined in order to further characterize this in vitro replication system; this system was devoid of any detectable extramitochondrial activity as determined by: a) the mt origin of the DNA polymerase activity as evidenced by studies using different templates and inhibitors, b) absence of chloroplast or nuclear DNA, glucose -6-P-dehydrogenase (known to be present only in the cytosol and chloroplasts) and photosynthetic pigments in the mitochondrial fraction and c) the ability of maize mt topoisomerase I to relax positively supercoiled DNA.
Daniell
H
H
Department of Botany and Microbiology, Auburn University, AL 36849-5407.
Zheng
D
D
Nielsen
B L
BL
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D019915:DNA Primase; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D013379:Substrate Specificity; D003313:Zea mays / Q000378:metabolism*
D008345:Manganese; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D004264:DNA Topoisomerases, Type I; D008274:Magnesium