Part 1- DNA Replication Flashcards
DNA Helix as a template
The DNA double helix acts as a template
for its own duplication.
. In this way, a double helical DNA
molecule can be copied
precisely.
The semiconservative nature of DNA replication.
In a round of replication, each of the two strands of DNA is used as a template for the formation of a complementary DNA strand.
The original strands therefore remain intact through many cell generations.
The chemistry of DNA synthesis.
DNA polymerase catalyzes the stepwise addition of a
deoxyribonucleotide to the 3ʹ-OH end of a polynucleotide chain, the growing primer strand that is paired to an existing template strand.
DNA Polymerase
The incorporation reaction is driven by a large, favourable free-energy change, caused by the release of pyrophosphate
and its subsequent hydrolysis to two
molecules of inorganic phosphate
Dissociation of pyrophosphate relaxes the polymerase,
allowing translocation of the DNA by one nucleotide so
the active site of the polymerase is ready to receive the
next deoxyribonucleoside triphosphate.
Proofreading by DNA polymerase
Exonucleolytic proofreading by DNA
polymerase during DNA replication.
In this example, a C is accidentally
incorporated at the growing 3ʹ-OH end of a DNA chain.
The part of DNA polymerase that
removes the mis incorporated
nucleotide is a specialized member of
a large class of enzymes, known as
exonucleases, that cleave nucleotides
one at a time from the ends of
polynucleotides
The structure of a DNA replication fork
Because both daughter
DNA strands are
polymerized in the 5ʹ-to-
3ʹ direction, the DNA
synthesized on the
lagging strand must be
made initially as a series
of short DNA molecules,
called Okazaki
fragments, named after
the scientist who
discovered them
Left, replication fork
with newly
synthesized DNA in
red and arrows
indicating the 5ʹ-to-
3ʹ direction of DNA
synthesis
Right, the same fork a short
time later. On the lagging
strand, the Okazaki fragments
are synthesized sequentially,
with those nearest the fork
being the most recently
made
Bacterial Replisome
The replisome is a large protein
complex that carries out DNA
replication, starting at the replication
origin. It contains several enzymatic
activities, such as helicase, primase and DNA polymerase and creates a
replication fork to duplicate both the
leading and lagging strand
During the initiation stage of DNA replication, double-stranded
DNA within the origin of replication (oriC) is melted through the
action of the initiator protein, DnaA, generating single-stranded
DNA substrates for replication
The primosome with the
DnaB helicase and DnaG
Primase is assembled
DNA Helicase
DNA helicases are essential during
DNA replication because they
separate double-stranded DNA into
single strands allowing each strand
to be copied
The “winding problem”
A) For a bacterial replication
fork moving at 500
nucleotides per second, the
parental DNA helix ahead of
the fork must rotate at 50
revolutions per second.
(B) If the ends of the DNA
double helix remain fixed (or
difficult to rotate), tension
builds up in front of the
replication fork as it becomes
overwound.
If the tension continues to build
up, the replication fork will
eventually stop because further
unwinding requires more energy
than the helicase can provide.
DNA Topoisomerase
a class of
enzymes that reduce
supercoiling in DNA by
breaking and re-joining
one or both strands of
the DNA molecule
RNA Primase and Synthesis
Unlike DNA polymerase, RNA
primase can start a new
polynucleotide chain by joining
two nucleoside triphosphates
together.
The primase synthesizes a
short polynucleotide in the 5ʹ-
to-3ʹ direction and then stops,
making the 3ʹ end of this
primer available for the DNA
polymerase.
Visualization of DNA replication
Stages in the replication of circular
DNA molecules have been
visualized by electron microscopy.
Replication of a circular
chromosome produces a structure
resembling the Greek letter theta,
θ, as both strands are replicated
simultaneously (new strands shown
in light red).
The electron micrographs show
images of plasmid DNA being
replicated from a single replication
origin
DNA Ligase
DNA Ligase enzyme seals a broken phosphodiester bond.
As shown, DNA ligase uses a molecule of ATP to activate the 5ʹ end at the nick (step 1) before forming the new bond (step 2).
In this way, the energetically unfavourable nick-sealing reaction is
driven by being coupled to the energetically favourable process of
ATP hydrolysis
Telomer Replication
(Eukaryotes)
The 3ʹ end of the parental DNA
strand is extended by RNA-
templated DNA synthesis; this
allows the incomplete daughter
DNA strand that is paired with it to
be extended in its 5ʹ direction.
This incomplete, lagging strand is
presumed to be completed by
DNA polymerase α, which carries a
DNA primase as one of its subunits
