Download The replication of DNA

The replication of DNA
Kornberg
Meselson and Stahl
Cairns
Okazaki
1957
1958
1963
1968
DNA Replication
The driving force for DNA synthesis.
The addition of a nucleotide to a
growing polynucleotide chain by
a phosphodiester bonds release
one molecule of pyrophosphate.
The free energy release from
this reaction is rather small.
Additional free energy is
provided by the rapid hydrolysis
of the pyrophosphate into two
phosphate groups by an enzyme
know as pyrophosphatase.
DNA synthesis reaction is
essentially irreversible.
DNA Replication
The mechanism of DNA polymerase
• DNA polymerase uses a single
active site to catalyze the addition
of any of the four dNTPs.
• DNA polymerase monitors the
ability of the incoming nucleotide
to form an A:T or G:C base pair;
incorrect base pairing leads to
drammatically lower rates of
nucleotide addition.
DNA Replication
The mechanism of DNA polymerase
.
• DNA
polymerases
show
an
impressive ability to distinguish
between rNTPs and dNTPs. This
discrimination is mediated by the
steric exclusion of rNTPs from the
active site; the nucleotide binding
pocket is to small to allow the
presence of a 2’-OH on the
incoming nucleotide. This space is
occupied by two “discriminator”
amino acids that make van der
Waals contact with the sugar ring.
DNA Replication
The mechanism of DNA polymerase
Three dimensional structure of DNA polymerase resemble a
right hand and the three domains are called the thumb,
fingers and palm.
DNA Replication
The mechanism of DNA polymerase
THE PALM DOMAIN
The palm domain contain the primary
elements of the catalytic site. This
region binds two divalent metal ions
(Mg2+ or Zn2+).
DNA Replication
The mechanism of DNA polymerase
THE PALM DOMAIN
One metal ion (A) reduce the affinity
of the 3’-OH for its hydrogen. This
generate a 3’O- ready for the
nucleophilic attack of the α-phosphate
of the incoming dNTPs.
The second metal ion (B) coordinates
the negative charges of the β- and γphosphate of the dNTP and stabilizes
the pyrophosphate produced.
DNA Replication
The mechanism of DNA polymerase
THE FINGERS DOMAIN
The fingers domain are
also important for
catalysis. Once a correct
base pair is formed
between the incoming
dNTP and the template,
the fingers domain
moves to enclose the
dNTP.
DNA Replication
The mechanism of DNA polymerase
THE FINGERS DOMAIN
The primary change is a 40°
rotation of the O- helix in the
finger domain.
• A tyrosine (aromatic amino
acid) makes stacking
interactions with the base
of the dNTP and
• two positively charged
residues, lysine and arginine,
stabilize the pyrophosphate.
DNA Replication
The mechanism of DNA polymerase
THE FINGERS DOMAIN
also associates with the
template region, leading to
a nearly 90° turn of the
phosphodiester backbone
between the first and the
second base of the
template.
This bend serves to expose only the first template base after
the primer at the catalytic site and avoids any confusion.
DNA Replication
The mechanism of DNA polymerase
THE THUMB DOMAIN
is not intimately involved in catalysis. It
interacts with DNA that has been mostly
recently synthesized.
This serve two purpose:
• It maintains the correct position of the
primer and the active site
• helps to maintain a strong association
between the DNA polymerase and its
substrate.
It binds a primer-template junction.
DNA Replication
The mechanism of DNA polymerase
THE PROOFREADING EXONUCLEASE
The palm domain also monitors the
base pairing of the most recently
added nucleotide.
If the recently added nucleotide are
correctly base-paired, the palm domain
makes extensive hydrogen bond
contact with base pairs in the minor
groove of the newly synthesized DNA.
Mismatched DNA in this region
interferes with the minor groove
contacts and dramatically slows
catalysis.
DNA Replication
The mechanism of DNA polymerase
THE PROOFREADING EXONUCLEASE
Proofreading of DNA synthesis is
mediated by exonucleases
present in the palm domain of the
DNA polymerase. These
exonucleases are capable of
degrading DNA starting from a 3’
DNA end.
When an incorrect nucleotide is
incorporated, the rate of DNA
synthesis is reduced because of the
incorrect positioning of the 3’-OH
DNA Replication
The mechanism of DNA polymerase
THE PROOFREADING EXONUCLEASE
In the presence of a mismatch
3’-end, the last 3-4
nucleotide became single
stranded. The exonuclease
active site has a ten fold higher
affinity for single stranded 3’
ends respect to the active site.
Once bound, the mismatched
nucleotide is removed and a
properly base paired is
reformed in the active site.
Proofreading exonuclease activity reduces the
error rate from 10-7 to 10-8 errors per base
pair.
DNA Replication
The mechanism of DNA polymerase
PROCESSIVITY
The degree of processivity of DNA polymerases is defined as the average
number of nucleotides added each time the enzyme binds a primer-template
junction.
• Each DNA polymerase has a characteristic processivity that can range from
only a few nucleotide to more than 50.000 bases added per binding event.
• Once bound addition of nucleotides is very fast. The fastest DNA
polymerases are capable of adding as many as 1000 nucleotides per second to
a primer strand.
• Increased processivity is facilitated by the ability of DNA polymerases to
slide along DNA template. Each time a nucleotide is added the DNA partially
release from the polymerase (H-bond with the minor groove are broken, but
the electrostatic interaction with the thumb are maintained). The DNA
rapidly rebinds to the polymerase in a position that is shifted by 1 bp.
DNA Replication
Sliding DNA clamps
Sliding DNA clamps are
proteins composed of
multiple identical
subunits that encircle the
DNA double helix and
also bind tightly to DNA
polymerases at
replication forks.
In absence of the sliding
clamp, DNA polymerase
dissociates from the
template DNA on average
once every 20-100 bp
synthesized.
DNA Replication
Sliding DNA clamps
In the presence of the
sliding clamp, the DNA
polymerase still
disengages its active site
from the 3’-OH
frequently, but the
association with the
sliding clamps prevents
polymerase from
diffusing away from
DNA.
Thus, sliding clamp increase
the processivity of the
DNA polymerase. These
proteins are present from
bacteria to human.
DNA Replication
Sliding DNA clamps loaders
Sliding clamp loaders are proteins
that catalyze the opening and
placement of sliding camp on
DNA. These enzyme couple ATP
binding and hydrolysis to the
placement of sliding clamp around
primer template junction, every
time that this junction is present in
the cell.
The clamp loaders also remove
the slide clamp from DNA once
all of the enzymes that interact
with them have completed their
function.
DNA Replication
• In general, DNA is replicated by:
– uncoiling of the helix
– strand separation by breaking of the
hydrogen bonds between the complementary
strands
– synthesis of two new strands by
complementary base pairing
Replication begins at a specific site in the DNA
called the origin of replication (ori)
DNA Replication
Semidiscontinuous DNA replication
Problem
DNA polymerases can only add nucleotides from 5′→3′
but, the two strands of the double helix are
antiparallel.
Solution
The major form of replication that occurs in nuclear
DNA (eukaryotes), some viruses (e.g. the papovavirus
SV40), and bacteria is called semidiscontinuous DNA
replication..
replication
DNA Replication
Semidiscontinuous DNA replication
• Two strands in the
double helix separate at
an origin of replication,
exposing bases to form a
replication bubble that
contains two replication
fork in opposite
direction.
• One strand is
synthesized continuously
from start to finish and
the other strand is
synthesized in short,
discontinuous fragments.
DNA Replication
Semidiscontinuous DNA replication
• Once primed, continuous replication is possible on the 3′→ 5′ template
strand (leading strand). Leading strand synthesis occurs in the same
direction as movement of the replication fork.
DNA Replication
Semidiscontinuous DNA replication
• Discontinuous replication occurs on the 5′→3′ template strand (lagging
strand). DNA is copied in short segments called Okazaki fragments
moving in the opposite direction to the replication fork. The lagging
strand requires the repetition of primer synthesis, elongation, primer
removal with gap filling and joining of Okazaki fragments.
DNA Replication
Semidiscontinuous DNA replication
• Despite this extra steps synthesis of both strands occurs concurrently
• Nucleotides are added to the leading and lagging strands at the
same time and rate by two DNA polymerases, one for each strand.
• Fundamental features of DNA replication are conserved from E. coli to
humans.
DNA Replication in Bacteria
• DNA replication is bidirectional from
the origin of replication
• DNA replication occurs in both
directions from the origin of replication
in the circular DNA found in most
bacteria.
DNA Replication in Bacteria
Initiation of replication
• An origin of replication is a
site on chromosomal DNA
where a bidirectional
replication fork initiates or
fires.
• Most bacteria have a single,
well-defined origin (e.g. oriC in
E. coli)
• Usually A-T rich.
DNA Replication in Bacteria
Initiation of replication
• In E. coli the initiator protein DnaA binds to oriC and recruits a
complex of two protein; the DNA helicase (DnaB) and helicase loader
(DnaC).
Three AA-T rich repeated elements that
are the site of initial DNA unwinding.
Five DnaA
DnaA-binding sites
The combination of all the protein that function at the replication
fork is referred to as the REPLISOME
DNA Replication in Bacteria
DNA polymerases in bacteria
E. coli has at least 5 DNA polymerases that are distinguished by their enzymatic
properties, subunits composition and abundance.
• DNA Pol III is the primary enzyme involved in the replication of the
chromosome. One subunits called Klenow fragment has 5’-3’ polymerase activity;
the other has a proofreading exonuclease activity 3’-5’
• DNA Pol I is specialized for the removal of the RNA primer and also has a
proofreading exonuclease.
• The remaining three DNA Pol are specialized for DNA repair and lack
proofreading activities.
DNA Replication in Bacteria
DNA polymerases in bacteria
In E. coli, the coordinate action of these polymerases is facilitated by
physically linking them together in a large multiprotein complex called the
DNA Pol III holoenzyme that confers very high processivity.
The DNA pol III holoenzyme includes two copies of the core DNA Pol
III and one copy of the five protein γ complex (the E. coli sliding camp
loader).
The γ complex includes
two copies of the τ
protein
DNA Replication in Bacteria
Initiation of replication
• To begin DNA replication, unwinding
enzymes called DNA helicases cause the
two parent DNA strands to unwind and
separate from one another at the origin of
replication to form two "Y"-shaped
replication forks.
• These replication forks are the actual site
of DNA copying
DNA Replication in Bacteria
DNA HELICASES
DNA helicases, catalyze
the separation of the two
strand of the double helix
breaking only the H-bonds
that hold the two strands
together.
DNA helicases are
hexameric proteins that
assume the shape of a
ring.
DNA helicases use the
energy coming from the
ATP hydrolysis to encircle
one of the two single
strands at the replication
fork.
DNA Replication in Bacteria
DNA HELICASES
Each subunit has a hairpin protein
loop that binds a phosphate of the
DNA backbone and its two adjacent
deoxyribose components.
The coordinated movement of these
protein hairpins can pull the ssDNA
through the central pore of the
helicase.
DNA Replication in Bacteria
DNA HELICASES
There are specialized mechanisms
that open the DNA helicase ring and
place it around the DNA before
forming the ring (initiation of
replication).
Each DNA helicases moves along
ssDNA in a defined direction
(POLARITY).This direction is define
according to the strand of DNA
bound. In case of a DNA helicases
that functions on the lagging
strand the polarity is 5’-> 3’;
On the leading strand 3’-> 5’ .
DNA Replication in Bacteria
ssDNAssDNA
-binding protein (SSB)
• Helix destabilizing proteins bind to the
single-stranded regions so the two
strands do not rejoin
Active E. coli SSB is composed of four identical
19 kDa subunits.
DNA Replication in Bacteria
ssDNAssDNA
-binding protein (SSB)
Single stranded
binding proteins
prevent
premature annealing,
to protect the
single-stranded DNA
from being digested
by nucleases, and to
remove secondary
structure from the
DNA to allow other
enzymes to function
effectively upon it.
DNA Replication in Bacteria
PRIMASE
DNA primase is a type
of RNA
polymerase which
creates a RNA primer
Primase catalyzes the
synthesis of a short RNA
segment complementary
to a ssDNA template.
DNA polymerases can
not initiate
the synthesis of
a DNA strand without an
initial RNA or
DNA primer (for
temporary DNA
elongation).
DNA Replication in Bacteria
PRIMASE
In bacteria, primase
binds to the DNA
helicase forming a
complex called
the primosome. Primase
is activated by DNA
helicase where it then
synthesizes a short
RNA primer
approximately 11
±1 nucleotides long, to
which
new nucleotides can be
added by DNA
polymerase.
DNA Replication in Bacteria
• The τ proteins interact with
DNA helicase and also bind
to both DNA polymerase.
• One DNA Pol III core
replicates the leading
strand and the other the
lagging strand.
• SSB coats the ssDNA
regions.
DNA Replication in Bacteria
• Periodically DNA primase
will associate with the
DNA helicase and
synthetize a new primer on
the lagging strand
template.
• Lagging strand Pol
complete the previous
Okazaki fragment.
DNA Replication in Bacteria
When the lagging strand
DNA Pol completes an
Okazaki fragment, it is
released from the sliding
clamp and the DNA.
DNA Replication in Bacteria
The recently primed
lagging strand is then a
target of the clamp
loader, which
assembles a new sliding
clamp at the primertemplate junction
DNA Replication in Bacteria
DNA Pol binds the sliding
clamp and begin to
synthesize a new Okazaki
fragment
This process continues around the circular genome until the replication
forks meet each other at the terminus to generate two daughter
molecules
DNA Replication in Bacteria
1. Unwinding
The enzyme helicase separates and unwinds the DNA double helix.
The separated strands are held apart by single-strand binding proteins,
forming a replication fork
DNA Replication in Bacteria
2. Priming the Leading Strand
Starting from a short RNA primer, DNA polymerase III assembles a
new complementary strand on each parental (template) strand.
The two strands are replicated in opposite directions, always 5' -> 3'.
DNA Replication in Bacteria
3. Building the Leading Strand
The strand that is polymerized toward the replication fork is called
the leading strand.
DNA polymerase III replicates the leading strand as a continuous strand
moving into (5' -> 3') the replication fork.
The RNA primer is removed and replaced with DNA by DNA polymerase I.
DNA Replication in Bacteria
4. Priming and Building the Lagging Strand
The lagging strand is synthesized in short pieces called Okazaki
fragments, each with an RNAprimer.
These fragments are joined together by DNA ligase, after DNA
polymerase I has removed the RNA primers.