ISOLATION AND CHARACTERIZATION OF AN IN-VITRO DNA-REPLICATION SYSTEM FROM MAIZE MITOCHONDRIA

An in vitro DNA replication system from maize mitochondria has been is olated 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 templat es and inhibitors were determined in order to further characterize thi s in vitro replication system; this system was devoid of any detectabl e extramitochondrial activity as determined by: a) the mt origin of th e DNA polymerase activity as evidenced by studies using different temp lates and inhibitors, b) absence of chloroplast or nuclear DNA, glucos e -6-P-dehydrogenase (known to be present only in the cytosol and chlo roplasts) and photosynthetic pigments in the mitochondrial fraction an d c) the ability of maize mt topoisomerase I to relax positively super coiled DNA. (C) 1995 Academic Press, Inc.