How is DNA replicated? Conservative, semiconservative or dispersive? Meselson and Stahl (1958) used heavy nitrogen (15N) to demonstrate that replication is semiconservative. This flash animation at "DNA from the Beginning" describes this experiment and some of Lederbergs experiments with DNA polymerase: A half DNA ladder is a template for copying the whole. Taylor (1957) used 3H-thymidine label and colchicine on bean root-tip cells to also demonstrate semiconservative replication also shows that chromosome is probably one long DNA molecule Cairns (1963) used autoradiography to show that in E. coli replication proceeded from one point (he saw theta structures) called the origin of replication One direction or two? Use of two labels demonstrated that replication was bidirectional in prokaryotes and eukaryotes (the later label showed up on both sides of the initial label, showing that new DNA was being made in two places, on either side of the starting point) there are some exceptions (Col E1 plasmid) Continuous, semidiscontinuous or discontinuous? Kornberg isolated a DNA polymerase and it only worked in the 5' to 3' direction Okazaki suggested that on at least one strand DNA synthesis must be discontinuous used labelling experiments to show that DNA was initially synthesized in small fragments (Okazaki fragments) if no DNA ligase was present these would never get combined into larger DNA fragments discontinuous only on the lagging strand - semidiscontinuous As DNA polymerase requires a free 3' end there must be a primer Tuneko Okazaki finally demonstrated that an RNA primer of 10-12 bp was at the end of Okazaki fragments Enzymology of DNA Replication Helicase - separates the DNA strands SSB, single-strand binding protein - keeps the strands apart Topoisomerases wind and unwind the DNA Gyrase nicks both strands and acts as a swivel Primosome - a protein complex that makes the RNA primer Replisome - carries out elongation of the DNA DNA polymerase III is the key enzyme two DNA pol III molecules per replisome so probably does both leading and lagging strands at the same time 400 nt/second Here is an interactive tutorial on the structure of the beta subunit of DNA polymerase III, you'll need the chime plugin to see this. DNA polymerase I replaces the RNA primer with DNA DNA ligase links the fragments together [ Biol 207 ] [ Bell ] [ CSU Chico ] [ Library ] This document is copyright of Jeff Bell Last Update: Friday, October 27, 2000