Replication of M13 Duplex DNA In Vitro

Abstract

Replication of M13 DNA proceeds through three consecutive stages: (I) conversion of the viral single-stranded DNA to the double-stranded replicative form (SS→RF), (II) multiplication of replicative forms (RF→RF), and (III) synthesis of progeny single strands (RF→SS). Stages II and III employ M13 duplex DNA as template and depend on the phage-encoded gene-II protein. The switch from double-strand to single-strand synthesis is brought about by the accumulation of M13 gene-V protein (see review by Ray 1977).

For a biochemical characterization of the enzymatic reactions involved in M13 DNA replication, appropriate in vitro systems are needed. The term “in vitro,” as understood here, refers to a situation where the permeability barrier of the cell has been destroyed and DNA synthesis has become dependent on exogenous nucleotides both as substrates and as energy supply. It is useful to make a distinction between cellular and cell-free in vitro systems. Cellular in vitro systems (nucleotide-permeable cells) still retain the in vivo concentration and spatial arrangement of the components of the replication apparatus. Cell-free systems are obtained after complete disruption of the cells and removal of cellular debris by high-speed centrifugation. Cellular systems may resemble the living state more closely, but they have the disadvantage that they are in general not permeable to proteins. On the other hand, cell-free systems can be fractionated and complemented with macromolecules but they are prone to experimental artifacts due to the disruption of the cell.

As far as M13 DNA replication is concerned, only the first stage, the SS→RF conversion,...