When does replication occur?
• Full blown replication only occurs
MBLG1001 lecture 10
Replication…
the once in a lifetime event!
The once in a lifetime process
•
•
•
•
This is a very tightly regulated process
It must be coordinated with cell division
The whole genome is faithfully copied.
The process must be as error free as
possible as this is the template for the next
generation.
The replication forks
once, just before cell division
• BUT the DNA template is constantly being
repaired.
• There are some 130 separate genes for
DNA repair in your genome which work
away in the background all the time.
The problems revisited:
• Replication is bi-directional. The theta
model.
• Bacterial DNA is a closed circle so it will
get tangled when it is unwound.
• DNA polymerases only work in one
direction and need primers
• The strands must be pulled apart and
unwound.
Summarising what we know:
oriC
• Initiation is tightly regulated at one site.
• 2 replication forks start in opposite
directions from this site
• Elongation is semi-conservative,
proved by the Messelson Stahl
experiment, carried out by DNA
polymerases
1
Initiation
• E coli has one defined site, oriC, origin of
replication on the chromosome or
replicator
• There is a sequence, 200 – 300 bp long
which is recognised by the initiator
protein DnaA
• 20 – 30 DnaA monomers bind to oriC in a
coordinated ATP-dependent (needs energy)
fashion and recruit DnaB, a helicase,
Summarising
In prokaryotes (bacteria)
• One site
• A specific sequence
• Specific proteins bind in a specific order
• Eukaryotes have multiple chromosomes
and multiple oriC sites.
Helicase or DnaB
Helicase or DnaB
• DnaB or helicase is a hexameric protein
(6 subunits) which unwinds double
stranded DNA. This is actually a whole
class of enzymes (12 types occur in E. coli
alone) present in all cells.
• It first arrives in the initiation complex and
is present throughout replication
• To replicate, repair or transcribe DNA you
need access to the middle of the double
helix, where the information is stored.
• Helicases break the h-bonds (not the
covalent phoshpodiester sugar phosphate
backbone) thus separating the two
strands. It requires energy to perform this
function, hydrolyzing NTPs to NDPs + Pi.
Once the strands are apart..
We need to stop them tangling..
• They need to be kept apart
• This is done by single stranded binding
protein (ssbp)
• Single stranded binding protein (ssbp)
prevents the two strands reannealing, as
well as forming intrastrand loops and
nuclease attack. The E. coli ssbp is a
tetramer which binds to the single
stranded DNA melted by helicase.
• This is particularly a problem with a closed
circle. As you unravel the 2 strands at one
end they will become increasingly tangled
at the other end.
• This is solved with a class of proteins
known as topoisomerses.
2
The tangling problem
• Topoisomerases. These enzymes
‘untangle’ the DNA in the regions still to be
copied.
• There are 2 types of topoisomerases; type
I and type II.
• Type I topoisomerases cut one strand of
the backbone, allow the other strand to
pass through the gap then reform.
Negative and positive
supercoiling
The tangling problem
• Type II Topoisomerases. DNA gyrase is
the most famous. Clark p107 Drugs used
• It cuts both strands and introduces
negative supercoiling to the DNA.
• It requires an energy source. (Type I
enzymes get their energy from the potential
energy stored in the stressed DNA)
• DNA is normally in a state of slight
negative supercoiling. Why?
We need a primer to get going..
Clark p89 Malacinski p39
• Negative supercoiling is when the 2
strands of DNA have twisting energy
applied in the opposite direction to the
twist of the helix. The double helix is then
easier to melt.
• Positive supercoiling is when the 2
strands have twisting energy applied in the
same direction as the helix. The strands
are more difficult to pull apart.
• Primase is an RNA polymerase and is
part of the primosome complex. It
produces short RNA primers (11-12 nt) for
DNA pol III at initiation and in the lagging
strand.
The elephant in the room
The elephant in the room!
• How do we get around the fact that the
replication fork is copying both strands
simultaneously but the enzyme only works
in one direction!
• Enter the leading and lagging strands.
• One strand is easy…this is the leading
strand. It is the parent strand in the
orientation 3’ to 5’
3
Replication Fork
Replication Fork
Direction of fork movement
DNA gyrase
Helicase
Primase
Replication Fork
Replication Fork
The 2
assemblies of
DNA pol III
are tethered
together
Single
stranded
binding
protein
DNA polymerase III subassembly
The leading strand
The leading strand
Parent strand
5’P
3’OH
dNTP
HO
Parent strand
5’P
5’P
3’OH
3’OH
• DNA polymerase simply works its way
around the chromosome on this strand
5’P
Newly synthesised strand
4
Newly
synthesised
primer
Replication Fork
The lagging strand
5’P
Newly synthesised DNA
• The template must wrap around and
approach the DNA pol III assembly from
the opposite direction
• It is synthesised discontinuously in short
stretches known as Okazaki fragments,
each with a new primer
• The primers are removed and the
fragments join up
RNA primer
3’OH
5’P
5’P
Previously
synthesised
primers
Replication Fork
The journey analogy
5’P
Newly synthesised DNA
5’P
RNA primer
Lagging
strand
synthesis
3’OH
5’P
dNTPs
3’OH
Leading
strand
• If replication is taking the equivalent of a
400 k journey in 40 min with an error every
170 k imagine this journey is taken by 2
delivery trucks
• One truck drives the route non-stop.
• The other truck stops every second and a
delivery person must take a detour off the
road before rejoining it again!
5’P
Processing the lagging strand
Sealing the nick….
• Once the Okazaki fragment has been
made DNA pol I comes in, removes the
primer (with its 5’ to 3’ exonuclease
activity) and fills in the gap (with its 5’ to 3’
polymerase activity).
• The nick is sealed by DNA ligase.
• DNA ligase seals nicks in the sugar
phosphate backbone, reforming the
phosphodiester bond.
• To do this it needs a source of energy. In
E. coli this is provided by NAD; in higher
organisms ATP is the energy source. DNA
pol I can’t join the nick as it relies on the
hydrolysis of pyrophosphate from dNTPs
to form the phosphodiester bond.
5
Gap sealed with DNA ligase
DNA pol I
Using its 5’ to
3’polymerase and its
proof reading 3’
exonuclease activity
5’P
3’
nucleotides
OH
5’
Using its 5’ to 3’
exonuclease
activity it will
chew up the
RNA primer
before it. This is
also known as its
intrinsic RNaseH
activity
Figure 28.10
General features of a replication fork. The DNA duplex is unwound by the action of DNA
gyrase and helicase, and the single strands are coated with SSB (ssDNA-binding protein).
Primase periodically primes synthesis on the lagging strand. Each half of the dimeric
replicative polymerase is a “core” polymerase bound to its template strand by a β-subunit
sliding clamp. DNA polymerase I and DNA ligase act downstream on the lagging strand to
remove RNA primers, replace them with DNA, and ligate the Okazaki fragments.
Figure 28.6 The semidiscontinuous model for DNA replication. Newly synthesized DNA is shown
as red. Because DNA polymerases only polymerize nucleotides 5 3, both strands must be
synthesized in the 5 3 direction. Thus, the copy of the parental 3 5 strand is
synthesized continuously; this newly made strand is designated the leading strand. (a) As the helix
unwinds, the other parental strand ( the 5 3, strand) is copied in a discontinuous fashion
through synthesis of a series of fragments 1000 to 2000 nucleotides in length, called the Okazaki
fragments;
the strand constructed from the
Okazaki fragments is called the
lagging strands. (b) Because both
strands are synthesized in
concert by a dimeric DNA
polymerase situated at the
replication fork, the 5 3
parental strand must warp around
in trombone fashion so that the
unit of the dimeric DNA
polymerase replicating it can
move along it in the 3 5
direction. This parental strand is
copied in a discontinuous fashion
because the DNA polymerase
must occasionally dissociate from
this strand and rejoin it further
along. The Okazaki fragments
are then covalently joined by DNA
ligase to form an uninterrupted
DNA strand.
6