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Nucleic Acids Research, 1982, Vol. 10, No. 18 5621-5635
© 1982

MOLECULAR BIOLOGY

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

Hans-Dieter Riedel, Herbert König, Hans Stahl and Rolf Knippers

Fakultät für Biologie, UniversiTät Konstanz D-7750 Konstanz, FRG

Received July 21, 1982. Revised August 31, 1982. Accepted August 31, 1982.

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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