DNA Replication
• during cell division in eukaryotic
cells, the replicated genetic
material is divided equally
between two daughter cells.
• it is important that each cell gets
an exact copy of the parent
cell’s DNA.
• in replicating, the DNA double
helix unwinds (H-bonds between
the strands are broken) and
each single strand acts as a
template for a new strand.
The Big Picture
Semi-Conservative Replication
•
DNA replicates semiconservatively (each
daughter strand contains
1 parent strand and 1
new strand).
• much of our
understanding of the
process has come from
studying prokaryotic
systems (like E. coli).
Starting Replication
• replication begins
when proteins -DNA
helicase, DNA
gyrase and single
stranded binding
proteins) bind to a
certain site on the
DNA called the
replication origin.
Replication Bubble
• the circular DNA in
prokaryotes will have 1
replication origin, while in
eukaryotes, there are
may be several of these
along a given stretch of
DNA.
DNAP III
• replication of the DNA
molecule proceeds in a 5’
to 3’ direction along each
strand, thus, in opposite
directions.
•
nucleotides are added by
an enzyme DNA
Polymerase III, which
adds dNTPs
(deoxyribonucleoside
triphosphates to a free 3’
end.
Adding Nucleotides
• DNA polymerase III
needs a free 3’ end to
begin, so another
enzyme, primase
lays down a 10-60
base pair stretch of
RNA primer to allow
DNAP III to start.
Adding dNTPs
• note that the dNTPs
contain 3
phosphates, while
the nucleotides only
contain 1.
• DNAP III breaks the
bond between
phosphates and uses
the energy released
to add the base to
the growing strand.
Leading vs. Lagging
• So, one strand (the
leading strand) is
synthesized continuously
in the 5’-3’ direction, while
the other (the lagging
strand) is synthesized
discontinuously in the 5’3’ direction.
• lagging strand
synthesis works by
having RNA primers
repeatedly added as the
replication fork moves
along.
Okazaki Fragments
• DNAP III forms small stretches
(100-200 base pairs long in
eukaryotes) of new sequence
called Okazaki fragments in
the 5’-3’ direction.
• DNA Polymerase I removes
the RNA primers from both the
leading and lagging strands
and replaces them with the
correct dNTPs.
• the Okazaki fragments are
joined together by another
enzyme DNA ligase by
creating a phosphodiester
bond.
• as the new strands are made,
two double stranded DNA
molecules automatically reform the double helix.
Quality Control
• because it is so important
to ensure that an exact
copy of the parent DNA
strand is made, both
DNAP I and III have a
proofreading capability
built in.
• both enzymes have
exonuclease activity,
that is they can remove a
nucleotide added in error
and replace it with the
correct one.
Overall….
http://www.youtube.com/watch?v=-mtLXpgjHL0