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Nucleic Acids Research, 1978, Vol. 5, No. 5 1689-1700
© 1978

Articles

A cell-free system for the replication of bacteriophage M-13 duplex DNA

Peter K. Schneck, Bob van Drop, Walter L. Staudenbauer and Peter Hans Hofschneider

Max-Planck-lnstitut für Biochemie D-8033 Martinsried bei München, GFR

Received March 13, 1978. Cell-free extracts from M-l3 am5 infected Escherichia coli cells which are highly concentrated on cellophane membrane disks replicate efficiently endogenous M-13 duplex DNA. If the reaction is carried out in the presence of bromodeoxyuridine triphosphate, the majority of the label is found in two classes of hybrid DNA molecules in which either the viral or the complementary strand is newly synthesized. A minor portion of the label is incorporated into fully synthetic duplex DNA. DNA synthesis requires ATP and is inhibited by nalidixic acid, novobiocin, and arabinosylnucleoside triphosphates. Rifampicin blocks preferentially the synthesis of molecules with labeled complementary strands. A similar effect is observed upon addition of the helix-destabilising M-13 gene V protein. In contrast, addition of E. coli helix-destabilising protein (Eco HD-protein) stimulates the synthesis of both types of hybrid DNA molecules as well as the formation of fully synthetic duplex DNA.


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