Chapter 6
Molecular
Biology of DNA
Replication and
Recombination
Jones and Bartlett Publishers © 2005
DNA Replication problems
• Start (origin) of replication
• Initiation sites
• Elongation – direction is important
• Accuracy
How does DNA replicate?
• Possibilities:
– Make two molecules, but with pieces mixed
up.
– Exact copy (conservative)
– Use each strand to make copy (semiconservative)
Replication of DNA by strand separation and copying of the two
template strands using A-T and G-C base pairing
(semi-conservative replication)
Experimental proof of semi-conservative
replication of DNA
Mehelson and Stahl experiment
Taylor, Woods and
Hughes experiment
Demonstrating
semiconservative
DNA replication
Vicia faba
Use of a thymidine analog (BUdR) provides
cytological proof that DNA in chromosomes also
replicates semi-conservatively
Replication of a circular DNA molecule
through a qstructure ( in a plasmid)
Replication can be uni- or bi-directional
During q-form replication, both parental DNA strands remain
intact. DNA replication begins at special sequences called
“origins of replication”. A single DNA molecule may have one
(E. coli chromosome) or many origins (a human chromosome
can have more than a thousand origins).
Replication of a circular DNA molecule
by a “rolling circle” mechanism
During rolling circle replication, one of the template DNA
strands is cut to create a primer 3’-OH end.
For every round of replication, the tail of the
rolling circle becomes one unit longer
One strand of
the rolling
circle grows
continuously
while the
other is made
in small
pieces
(Okazaki
fragments).
This is
common in
phages.
Replication of a linear eukaryotic chromosome
Speed of replication
• E. coli and other prokaryotes have faster
replication than eukaryotes: 1500 bp per
second. (30 minutes, but 20 min. gen. time).
• Drosophila has multiple origins of
replication, and can make 50 nucleotide
pairs per second.
• Typical eukaryotes have origins every
40,000 bp, and can replicate all
chromosomes in 5-10 hours.
First Enzyme Found to Be Involved in
DNA Replication
• DNA polymerase III – covalent addition
of nucleotides to the DNA chain.
• Requirements: deoxynucleoside
triphosphates, Mg++ ions, template DNA,
and a primer
DNA Polymerase Characteristics
• Absolute requirement for a template
strand, and new base selection is template
driven.
• Polymerization is always in the 5’  3’
direction on the new strand, antiparallel
to the template strand.
• Initiation of new chain growth is not
possible without a primer with a 3’ OH
Other Enzymes
• DNA helicase – unwinds DNA
• DNA gyrase – relaxes supercoils
– Also called topoisomerase II
• primase – synthesizes RNA primer, 5-12
bp long
• 5’  3’ exonuclease – removal of primer
• DNA ligase – joins ends of DNA
Model of an E. coli DNA replication fork
showing the many proteins that play a role there
Prevention of knotting of DNA (as strands are
separated) by DNA gyrase working ahead of the
DNA replication fork
DNA Replication
The components that are different in
RNA relative to DNA
Priming of DNA synthesis with an RNA segment
primosome polymerase alpha, 1520 other polypeptides
make ~12 nucleotides
of RNA, then ~23
DNA nucleotides.
New DNA chains are initiated by
short RNA primers
Addition of a deoxynucleotide
to the 3’-OH end of a primer chain
A misinserted deoxynucleotide is excised by the
proofreading exonuclease function
of DNA polymerase
One side of the fork grows continuously (leading side)
while the other side grows by making small
DNA pieces (lagging side)
Sequence of events in the joining of adjacent
precursor fragments in eukaryotes
Accuracy of replication
• If human DNA error rates were one in a
105 base pairs, there would be 60,000
mistakes for each cell division cycle.
• The actual rate is 1 in 10 billion, or about
one mistake for every three cell divisions.
• 3’ –to- 5’ exonuclease activity of DNA
polymerase results in proofreading.