Abstract
A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.
Highlights
Previous work in this laboratory has resulted in the isolation of a transcription activity from human mitochondria [5]
Fraction I1 was immediately applied to a 1.5 X 10-cm column of phosphocellulose that had been equilibrated in buffer A containing 0.3 M NaCl
A nuclease activity was present among the mitochondrial proteins and may account for the generation of 3'-hydroxyl described for assaying M13 DNA replication except that [(u-~*P]GTPends that results in significant levels of rNTP-independent
Summary
Characterization of a fraction of mitochondrial proteins, isolated from human tissue culture cells, that can initiate replication on single-stranded DNA templates. Assay of DNA P a l y ~ e r y~ Ae ctivity-Each reaction mixture was in a total volume of 25 pl and contained 20 mM Tris.HC1, pH 7.5, 0.5 mMMnC12, BSA at 0.1 mg/ml, 50 mM sodium phosphate, 0.1 M NaCl, polyA'oligo(dT) a t 25pg/ml, 10 p~ [ C ~ - ~ ~ P J (T8,T00P0 to 15,000cpm/pmol), 1mM 2-mercaptoethanol and varying amounts of mitochondrial enzymes. A nuclease activity was present among the mitochondrial proteins and may account for the generation of 3'-hydroxyl described for assaying M13 DNA replication except that [(u-~*P]GTPends that results in significant levels of rNTP-independent ( 1 5 0 , ~cpm/pmol) was used at 4 p~ asthe only radiolabeled DNA synthesis with either template. The reaction mixtures contained 0.4 unit of E. coli DNA polymerase I large fragment
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