Watson and Crick hypothesized that DNA replication is semiconservative - but they
also had to consider the conservative and dispersive modes
of replication as alternative hypotheses.
Meselson-Stahl tested this hypothesis by growing DNA in a medium with heavy
nitrogen isotope (15N) - then removing it and placing it in a
light nitrogen medium (14N) - then took samples and subjected them to equilibrium
density-gradient centrifugation in cesium chloride
solution at different times:
- If conservative, there should always be a heavy band - light band starts with
the same amount, then increases everytime.
- If dispersive, first only hybrid band - then the whole band starts moving up
towards the light region while increasing.
- If semiconservative, hybrid band should always be there and light band
starts to increase from 0.
Taylor showed that semiconservative replication also occur in eukaryotes. Cells
were put in medium with bromodeoxyuridine (BrdU) that is
incorporated into DNA in place of thymidine and could be contrasted with others
after staining. After 2 generations, he found that one
chromatid of each chromosome was composed of 2 BrdU-containing strands,
whereas the other chromatid was a hybrid.
Separation of DNA molecules creates topological problems (torsional stress). An
overwound DNA becomes positively supercoiled ahead of the
www.notesolution.com fork. DNA gyrase (type II topoisomerase) relieves the mechanical strain during
replication - they travel along the DNA ahead of the
replication fork and removes positive supercoiling by cleaving and sealing.
DNA must have specific structural requirements to initiate replication. DNA
polymerase cannot initiate the formation of DNA strand - it can
only add nucleotides to a 3’ hydroxyl terminus of an existing strand. The strand that
provides this is called a primer.
DNA polymerase III is the major enzyme responsible for replication, but there’s only
10 copies for every 300-400 polymerase I.
New DNA strand are always synthesized 5’-3’ direction. So, there’s leading and
lagging strand. Replication is semidiscontinuous.
Okazaki found that if he incubated bacteria in 3H thymine, after a few seconds most
of the labeled DNA was short, but after a few minutes
most of it was large. This suggested that a portion of the DNA was constructed in
small segments and then joined by DNA ligase.
Initiation is accomplished by a type of RNA polymerase called primase, which
constructs a short RNA primer required for initiation.
At the replication fork, DNA helicase uses ATP to unwind DNA by moving along one
of the DNA strands and breaking hydrogen bonds.
The major one is DnaB, which first binds to origin of replication by the help of
DnaC, and then proceeds. DNA unwinding is aided by the
www.notesolution.com attachment of single stranded DNA-binding (SSB) proteins to the separated DNA
strands to prevent them from rewinding.
In bacteria, primase and helicase associate together to form primosome.
The same DNA polymerase III synthesizes all lagging strands. It moves along with
the DNA polymerase III that is synthesizing the leading
strand - they are both a part of a single protein complex. This is accomplished by
causing the lagging strand to form a loop, so that
both would still be synthesizing in the 5’-3’ direction even though thy are moving
together along the DNA.
DNA polymerase III is a part of DNA polymerase III holoenzyme (or replisome).
Another component is b clamp - keeps polymerase associated
with the DNA template. The assembly of b clamp with the DNA molecules requires
a clamp loader, which is another part of the replisome.
Once the DNA enters the b clamp, the ATP on the loader is hydrolyzed and and
openning of the clamp closes.
When DNA polymerase synthesizes an Okazaki fragment, it disengages from the b
clamp and binds to another one.
DNA polymerase I is responsible for repairing and replacing RNA primers on
Okazaki fragments with DNA. It has 3’-5’ and 5’-3’ exonucleases,
in addition to its polymerizing activity. The 5’-3’ exonuclease removes RNA primers
and its polymerizing activity adds DNA in its place.
www.notesolution.com If the incoming nucleotide does not form the proper geometry with the other
nucleotide then the fingers of DNA polymerase I cannot fold
onto the palm, and so, cannot catalyse the reaction to link the two (conformational
change is required for the enzymatic activity). When