DNA Replication

Overview

This module looks at the way DNA reproduces itself, which is one of the necessary properties for its role as hereditary material.

Objectives

1. Understand the semiconservative nature of DNA replication. Realize that the process begins at unique origins of replication, and proceeds bidirectionally.
2. Know that DNA synthesis is catalyzed by a family of enzymes called DNA polymerases. Understand that DNA polymerase has a requirement for a template on which to synthesize the new DNA strand, and for a primer from which to extend the DNA strand.
3. Understand the various functions of the RNA polymerases, such as exonuclease and polymerase activities, and their function in the replication process.
4. Know the other components of the replication apparatus, and their basic functions. Understand the principles of leading strand synthesis and lagging strand synthesis.
5. Realize that eukaryotes require the activity of telomerase to complete the synthesis of their linear chromosomes.

The Semiconservative Nature of DNA Replication

One property of the genetic material necessary for its function is the ability to replicate (reproduce) itself. After it was established that DNA is the genetic material, attention turned toward how DNA was replicating in living organisms.

The Watson-Crick model of DNA structure (as outlined in the module on nucleic acids) suggested a possible mechanism for replication of DNA molecules. The nature of base pairing meant that if the two strands of a DNA molecule were separated, they could each serve as a template for the creation of a complementary strand by bringing in individual nucleotides to base pair with their complementary base on the template, and joining the new nucleotides together. Thus, each DNA molecule after replication would consist of one of the original strands plus one newly synthesized strand. This model of DNA replication is called semiconservative.

Semiconservative was not the only model of DNA replication, however. Other proposed models included conservative replication and dispersive replication. Conservative replication proposed that after replication, one DNA molecule consists entirely of newly synthesized DNA whereas the other molecule is entirely original DNA. Dispersive replication suggested that each DNA molecule after replication might consist of segments of new and old DNA interspersed. It would be difficult to devise a mechanism by which this latter outcome might occur, but until evidence to the contrary was produced, it had to be considered. The three possible models of DNA replication are depicted below.

To distinguish between these possibilities, Meselson and Stahl did the following experiment:

First, they grew bacteria for many generations in a growth medium containing ¹⁵N. This is a heavy isotope of nitrogen (in contrast to the normal isotope, ¹⁴ N), which over many generations would be incorporated into all nitrogen-containing molecules of the cells, including DNA. DNA isolated from these cells could be distinguished from normal DNA because it would have a higher density.

The bacteria grown in heavy nitrogen were then transferred to growth medium containing ¹⁴ N for one round of replication. This lighter isotope would incorporate into any newly synthesized DNA. If semiconservative replication occurred, then each DNA molecule after replication would contain heavy nitrogen and light nitrogen, and would therefore have a density intermediate between the two. Conservative replication would produce one DNA molecule containing heavy nitrogen and one molecule containing light nitrogen, so there would be two different densities. Dispersive replication would produce a single intermediate density, just like semiconservative.

The observed density of the DNA after one round of replication was intermediate. Replication was therefore either semiconservative or dispersive. These possibilities could be distinguished after a second round of replication. After two rounds, semiconservative replication would produce two DNA molecules containing only light nitrogen, and two DNA molecules containing one light strand and one heavy strand. Therefore there would be two different densities: light and intermediate. Two rounds of dispersive replication would produce four DNA molecules, each of which would contain mostly light nitrogen and some heavy nitrogen. There would be a single density (we'll call it 'slightly heavy'). When density of the DNA was measured after two rounds, two densities were observed: light and intermediate, indicating that DNA replication is semiconservative, and not dispersive or conservative.