Watson and Crick were particularly excited about their model because
the complementary nature of the DNA molecule suggested a way in which
it might self-replicate. The two strands could separate from one
another, each still containing the complete information, and
synthesize a new strand. However, experiments still had to be done to
prove that this model was true:
Messelson and Stahl
In 1957, Matthew Meselson and Franklin Stahl did an experiment to
determine which of the following models best represented DNA
- Did the two strands unwind and each act as a template for new
strands? This is semiconservative replication, because each new
strand is half comprised of molecules from the old strand.
- Did the strands not unwind, but somehow generate a new double
stranded DNA copy of entirely new molecules? This is
In order to determine which of these models was true, the
following experiment was performed: The original DNA strand was
labelled with the heavy isotope of nitrogen, N-15. This DNA was
allowed to go through one round of replication with N-14, and then the
mixture was centrifuged so that the heavier DNA would form a band
lower in the tube, and the intermediate (one N-15 strand and one N-14
strand) and light DNA (all N-14) would appear as a band higher in the
tube. The expected results for each model were:
The actual results were as expected for the semiconservative model and
thus Watson and Crick's suspicion was borne out.
Biochemical Mechanism of DNA Replication
It is very important to know that DNA replication is not a passive and
spontaneous process. Many enzymes are required to unwind the double
helix and to synthesize a new strand of DNA. We will approach the
study of the moelcular mechanism of DNA replication from the point of
view of the machinery that is required to accomplish it. The unwound
helix, with each strand being synthesized into a new double helix, is
called the replication fork.
The Enzymes of DNA Replication
- Topoisomerase is responsible for initiation of the
unwinding of the DNA. The tension holding the helix in its coiled and
supercoiled structure can be broken by nicking a single strand of DNA.
Try this with string. Twist two strings together, holding both the top
and the bottom. If you cut only one of the two strings, the tension of
the twisting is released and the strings untwist.
- Helicase accomplishes unwinding of the original double
strand, once supercoiling has been eliminated by the topoisomerase.
The two strands very much want to bind together because of their
hydrogen bonding affinity for each other, so the helicase activity
requires energy (in the form of ATP ) to break the strands apart.
- DNA polymerase proceeds along a single-stranded molecule of
DNA, recruiting free dNTP's (deoxy-nucleotide-triphosphates) to
hydrogen bond with their appropriate complementary dNTP on the single
strand (A with T and G with C), and to form a covalent phosphodiester
bond with the previous nucleotide of the same strand. The energy
stored in the triphosphate is used to covalently bind each new
nucleotide to the growing second strand. There are different forms of
DNA polymerase , but it is DNA polymerase III that is responsible for
the processive synthesis of new DNA strands. DNA polymerase cannot
start synthesizing de novo on a bare single strand. It needs a
primer with a 3'OH group onto which it can attach a dNTP. DNA
polymerase is actually an aggregate of several different protein
subunits, so it is often called a holoenzyme. The holoenzyme
also has proofreading activities, so that it can make sure that it
inserted the right base, and nuclease (excision of nucleotides)
activities so that it can cut away any mistakes it might have made.
- Primase is actually part of an aggregate of proteins called
the primeosome. This enzyme attaches a small RNA primer to the
single-stranded DNA to act as a substitute 3'OH for DNA polymerase to
begin synthesizing from. This RNA primer is eventually removed by
RNase H and the gap is filled in by DNA polymerase I.
- Ligase can catalyze the formation of a phosphodiester
bond given an unattached but adjacent 3'OH and 5'phosphate. This can
fill in the unattached gap left when the RNA primer is removed and
filled in. The DNA polymerase can organize the bond on the 5' end of
the primer, but ligase is needed to make the bond on the 3' end.
- Single-stranded binding proteins are important to maintain
the stability of the replication fork. Single-stranded DNA is very
labile, or unstable, so these proteins bind to it while it remains
single straded and keep it from being degraded.
The Replication Fork
Why can DNA polymerase only act from 5' to 3'? The reason is the
relative stability of each end of DNA. A triphosphate is required to
provide energy for the bond between a newly attached nucleotide and
the growing DNA strand. However, this triphosphate is very unstable and
can easily break into a monophosphate and an inorganic pyrophosphate,
which floats away into cell. At the 5' end of the DNA, this
triphosphate can easily break, so if a strand has been sitting in the
cell for a while, it would not be able to attach new nucleotides to
the 5' end once the phosphate had broken off. On the other hand, the
3' end only has a hydroxyl group, so as long as new nucleotide
triphosphate are always brought by DNA polymerase, synthesis of a new
strand can continue no matter how long the 3' end has remained free.
This presents a problem, since one strand of the double helix is
5' to 3' , and the other one is 3' to 5'. How can DNA polymerase
synthesize new copies of the 5' to 3' strand, if it can only travel in
one direction? This strand is called the lagging strand, and
DNA polymerase makes a second copy of this strand in spurts, called
Okazaki fragments, as shown in the diagram. The other strand
can proceed with synthesis directly, from 5' to 3', as the helix
unwinds. This is the leading strand.