Lecture 12: DNA Replication
Three Postulated Methods of DNA Replication
The model of replication proposed by Watson and Crick
The helicase unwinds and each strand serves as a template for a new strand.
The new strands synthesized are identical to the parental strands.
The two strands of the original molecule serve as templates for the two
strands of a new DNA molecule, and then rewind into an “old” molecule.
The two copies separate from their templates to wind into the “new”
Neither parental strand is conserved
Both chains contain old and new segments.
The Semi-Conservative Model
1958 – Meselson and Stahl proved in an experiment that DNA is replicated in
a semi-conservative nature.
In their experiment they had to distinguish between old parental DNA and
newly replicated DNA
They did this by using “heavy” nitrogen isotope to tag parental strands 15N is
heavy because it has one more neutron in its nucleus.
1.) Ecoli was grown in the heavy 15N medium for several generations, where
it was incorporated into the nitrogenous bases of DNA
2.) Then they transferred the bacteria to a culture medium containing the
normal light isotope 14N where the bacteria grew and divided for several
3.) The DNA samples were extracted into CsCl and mixed at a high rate in the
centrifuge. This created a density gradient so that molecules densities
separated in to bands. The densest settled closer to the bottom of the tube.
The predicated banding patterns matched the semi-conservative model
Major steps in DNA replication:
1. The two strands of DNA unwind for replication to occur
2. Nucleotides are added only to the existing chain
3. The overall direction of new synthesis is in the 5’ to 3’ direction,
which is a direction antiparallel to the template strand.
4. Nucleotides enter into a newly synthesized chain according to the
A-T and G-C complimentary base-pairing rules
These are the main steps that require DNA replication. They are catalyzed by
a bunch of enzymes:
DNA Helicase: Catalyzes the unwinding of the double helix, which produces a
Replication Fork: Opens slowly and continually. A Y shape form the helical
DNA and unwound template strands.
o Synthesis occurs as soon as the nucleotides are exposed.
o Synthesis occurs in the 5’ to 3’ orientation
Single Stranded Binding Proteins: Bind to the unwound template strands to
stabilize the DNA for the replication process
Primase: Provides a short nucleotide chain, called a primer, (which is made
of RNA instead of DNA) onto the DNA, because DNA polymerase can only
synthesize nucleotides onto an existing strand. (It can’t start from nothing)
DNA polymerase 3: assembles new DNA to the 3’ end of RNA primers.
o Can only assemble DNA in a 3’ to 5’ direction
o Only one of the DNA strands runs in this direction because they are
antiparallel to each other.
The Leading Strand: Runs 5’ to 3’ and is synthesized continuously
The Lagging Strand: Runs 3’ to 5’ and is synthesized discontinuously
o In discontinuous replication the polymerases add short sections –
called “Okazaki fragments” – of new nucleotides in the opposite
direction the DNA unwinds DNA polymerase 1: Removes the RNA primers and replaces with DNA, but
the fragments are not covalently joined, there is a ‘nick’ between them
DNA Ligase: seals the nick after RNA primers are replaced with DNA so the
lagging strand becomes a continuous polynucleotide sequence.
Topoisomerases: Remove over twisting and strain of DNA ahead of
replication fork in a circular DNA
Note – DNA replication is fast. So although process is show step by step spread out,
all enzymes actually operate at the replication fork.