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Circular single stranded phage M13-DNA as a template for DNA synthesis in protein extracts from Xenopus laevis eggs: evidence for a eukaryotic DNA priming activity.
Riedel HD
,
König H
,
Stahl H
,
Knippers R
.
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Unfractionated protein extracts from activated Xenopus laevis eggs contain all functions required for the chain elongation reactions in replicative DNA synthesis (A.Richter, B. Otto and R.Knippers, 1981, Nucl.Ac.Res. 9, 3793-3807). In order to further explore the DNA synthesizing capacity of this in vitro system and to obtain information on the DNA priming activity in these extracts single stranded phage M13-DNA was used as template for in vitro DNA synthesis. The main results of this investigation are: (i) single stranded circular template DNA is converted to a double stranded DNA form in an alpha-amanitin-insensitive reaction which is absolutely dependent on ribonucleoside triphosphates; (ii) the in vitro synthesized complementary strands are DNA fragments of 1000-2000 nucleotides lengths; (iii) the DNA primase activity copurifies through several column steps and sucrose gradient centrifugation with a DNA polymerase alpha. These activities may therefore be closely associated in a quarternary enzyme complex.
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