Chapter 10 – DNA Replication
Different Theoretical Modes of Replication
• Semiconservative replication has each newly replicated DNA molecule consisting of one “old”
strand and one “new” strand.
• Conservative replication will have two DNA molecules, one molecule with both strands
composed of “old” DNA and one molecule with both strands of “new” DNA
• Dispersive replication has both strands composed of a mixture of “old” and “new” DNA
Proof for Mode of DNA Replication
• The Meselson-Stahl Experiment
– Grew bacterial cells (E. coli) for many generations in medium supplemented with
15NH 4l as the only nitrogen source.
• Remember the nitrogenous bases have nitrogen atoms, so the DNA will now be
labelled15ith this “heavy” version of nitrogen 14
– N contains one more neutron than naturally occurring N.
– Molecules containing N are more dense than molecules containing N 14
– Importantly,14NA containing N can be distinguished from DNA
Sedimentation Equilibrium Centrifugation
• Samples forced by centrifugation into a density gradient of a heavy metal salt solution
– Usually CsCl (cesium chloride)
• Molecules of DNA will migrate into the density gradient.
– 15e DNA will stop moving when it reaches a region of CsCl that m14ches its own density
– N-DNA will move further down the tube of CsCl compared to N-DNA.
Semiconservative Replication in Eukaryotes
• In 1957 Taylor, Woods and Hughes provided evidence of semiconservative replication in broad
bean plants (Vicia faba).
• Technique used was autoradiography
– Can locate location of radioactive isotope in a cell
– A photographic emulsion is placed over a histological section/sample and placed in the
• Radioactivity exposes the emulsion just like light would expose a photographic
• Exposure of emulsion results in tiny black dots
– Locations of black dots = location of radioactive isotope in cell
Taylor et al. Experiment
• Labelled DNA with H thymidine for one generation
– Then placed in “cold” medium
– Arrested cells at metaphase with colchicine over two division cycles
– Performed autoradiography.
– Results, at first metaphase, both chromatids labelled, while in second metaphase only
one chromatid labelled ...semiconservative replication Origins, Forks and Units of Replication
• Origin of Replication
– Location where DNA replication is initiated
– Replication is bidirectional from the origin
• Replication Fork is where DNA strands are being unwound
– Initially appear at origin and then 2 forks move apart in opposite directions
• A replicon represents how much DNA is replicated following one initiation event
– Eg E. coli has only one origin of replication (oriC) for its entire 4.6 million base pairs (bp)
of circular DNA
DNA Polymerase I
• Kornberg accomplished in vitro synthesis of E. coli DNA. His reaction mixture included:
a. A DNA fragment (act as template).
b. Radioactively labeled dNTPs (dATP, dGTP, dTTP, and dCTP).
a. DNA Polymerase I (DNA Pol I).
b. Enzyme originally called the Kornberg enzyme, now known as DNA polymerase I. Once
isolated, its activity could be characterized, showing that the above components are
required, along with Mg ions for maximum activity.
Roles of DNA Polymerases
All DNA polymerases link dNTPs into DNA chains. Main features of the reaction:
– An incoming nucleotide is attached by its 5’-phosphate group to the 3’-OH of the
growing DNA chain. Energy comes from the release of two phosphates from the dNTP.
The DNA chain acts as a primer for the reaction.
– The incoming nucleotide is selected by its ability to hydrogen bond with the
complementary base in the template strand. The process is fast and accurate.
– DNA polymerases synthesize only from 5’ to 3’. Initiation of Replication in Prokaryotes
• Initiator proteins attach. E. coli’s initiator protein i