Physical studies on the replication of DNA in vitro

Density gradient analysis shows two stages in synthesis : molecules of hybrid density are synthesized in the first stage and a product free from primer is made in the second stage, and these results do not support a dispersive mechanism for replication.

Abstract

The synthetic DNA copolymers dAT‡ and dABU¯ have been used to study the replication of primer DNA by the DNA-synthesizing enzyme from E. coli. dAT was used as primer for the synthesis of dABU¯ and vice versa. The two copolymers are well separated by density gradient centrifugation and also give separate melting curves in low salt. Density gradient analysis shows two stages in synthesis : molecules of hybrid density are synthesized in the first stage and a product free from primer is made in the second stage. These results do not support a dispersive mechanism for replication. Synthesis begins promptly on most of the primer molecules and proceeds gradually, so that intermediate stages in the synthesis of new molecules are observed. Optical density melting curves provide strong support for a semi-conservative mechanism of replication. Hybrid base-pairs are formed in the first stage of synthesis, as shown by the presence of a hybrid melting zone, and product base-pairs are formed in the second stage. When dAT is used as primer for the synthesis of a small amount of dABU¯ (5%), more than half the primer is prevented from melting until the hybrid melting zone is reached. This suggests that small amounts of dABU¯ are added covalently at the growing ends of dAT strands, forming dAT : dABU¯ base-pairs at the ends of the dAT : dAT helix, and that melting begins at the ends of strands.