DNA Replication
II- DNA Replication
• Origins of replication
1. Replication Forks: hundreds of Y-shaped
regions of replicating DNA molecules
where new strands are growing.
5’
3’
Parental DNA Molecule
Replication
Fork
3’
5’
• Origins of replication
2. Replication Bubbles:
a.
Hundreds of replicating bubbles
(Eukaryotes).
b.
Single replication fork (bacteria).
Bubbles
Bubbles
Required components for the DNA replication:
1.
dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates),
(sugar-base + 3 phosphates)
2.
DNA template
3.
DNA polymerase
4.
Mg 2+ (optimizes DNA polymerase activity)
• Strand Separation:
1.
Helicase: enzyme which catalyze the
unwinding and separation (breaking HBonds) of
the parental double helix.
2.
Single-Strand Binding Proteins:
proteins
which attach and help keep the separated
strands
apart.
3.
Topoisomerase: enzyme which relieves
stress on the DNA
molecule by allowing
free rotation around a single strand.
Enzyme
DNA
Enzyme
• Priming:
1.
RNA primers: before new DNA strands can
form, there must be small
preexisting
primers (RNA) present to start the addition of new
nucleotides (DNA Polymerase).
2.
Primase: enzyme that polymerizes (synthesizes) the RNA Primer.
Three main features of the DNA synthesis reaction:
•
DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the
deoxyribose (on the last nucleotide) and
•
the 5’-phosphate of the dNTP.
Energy for this reaction is derived from the release of two of the three phosphates of
the dNTP.
•
DNA polymerase “ finds ” the correct complementary dNTP at each step in the
lengthening process.
•
rate ≤ 800 dNTPs/second
•
low error rate
•
Direction of synthesis is 5’ to 3’
DNA elongation:
Origin of replication (e.g., the prokaryote example):
• Begins with double-helix denaturing into single-strands thus exposing the bases.
• Exposes a replication bubble from which replication proceeds in both directions.
Initiation of replication, major elements:

Segments of DNA are called template strands.

Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.

Initiator proteins and DNA helicase binds to the DNA at the replication fork and
untwist the DNA using energy derived from ATP (adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA helicase)


DNA primase next binds to helicase producing a complex called a primosome
(primase is required for synthesis),

Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA
polymerase III adds nucleotides.

Polymerase III adds nucleotides 5’ to 3’ on both strands beginning at the RNA
primer.

The RNA primer is removed and replaced with DNA by polymerase I, and the gap is
sealed with DNA ligase.

Single-stranded DNA-binding (SSB) proteins (>200) stabilize the single-stranded
template DNA during the process.
• Synthesis of the new DNA Strands:
1. DNA Polymerase: with a RNA primer in
place,
DNA Polymerase (enzyme) catalyze
the
synthesis of a new DNA strand in the 5’ to 3’
direction.
5’
3’
Nucleotide
DNA Polymerase
RNA
Primer
5’
1- Leading Strand: synthesized as a single polymer in the 5’ to 3’ direction.
5’
3’
5’
Nucleotides
DNA Polymerase
RNA
Primer
2- Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously
.
DNA
Okazaki Fragment
Polymerase
RNA
Primer
5’
3’
3’
5’
Lagging Strand
DNA replication is continuous on the leading strand and semidiscontinuous on the
lagging strand:
Unwinding of any single DNA replication fork proceeds in one direction.
The two DNA strands are of opposite polarity, and DNA polymerases only synthesize
DNA 5’ to 3’.
Solution: DNA is made in opposite directions on each template.
•Leading strand
•Lagging strand
synthesized 5’ to 3’ in the direction of
the replication fork movement.
continuous.
requires a single RNA primer
synthesized 5’ to 3’ in the opposite
direction. semidiscontinuous (i.e., not continuous)
requires many RNA primers , DNA is
synthesized in short fragments.
5’
SSB Proteins
Okazaki Fragments
1
Polymerase III
2
3
Lagging strand
ATP
Helicase
+
Initiator Proteins
3’
primase
Polymerase III
base pairs
5’
RNA primer replaced by polymerase I
& gap is sealed by ligase
Leading strand
RNA Primer
3’
Model of DNA Replication
5. DNA ligase: a linking enzyme that
catalyzes the formation of a covalent bond
from the 3’ to 5’ end of joining stands.
Example: joining two Okazaki fragments together.
DNA ligase
5’
3’
Okazaki Fragment 1
Lagging Strand
Okazaki Fragment 2
3’
5’
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond
DNA replication proteins
Enzyme
Function in DNA replication
DNA Helicase
Also known as helix destabilizing enzyme. Unwinds the DNA
double helix at the Replication Fork.
DNA Polymerase
Builds a new duplex DNA strand by adding nucleotides in the
5' to 3' direction. Also performs proof-reading and error
correction. There exist many different types of DNA
Polymerase, each of which perform different functions in
different types of cells.
(RNA Polymerase)
Primase
Provides a starting RNA short primers for DNA polymerase
to begin synthesis of the new DNA strand.
DNA clamp
A protein which prevents elongating DNA polymerases from
dissociating from the DNA parent strand.
Single-Strand Binding
(SSB) Proteins
Bind to ssDNA and prevent the DNA double helix from
reannealing after DNA helicase unwinds it, thus maintaining
the strand separation, and facilitating the synthesis of the
nascent strand.
Topoisomerase
Relaxes the DNA from its super-coiled nature.
DNA Ligase
Re-anneals the s and and joins Okazaki Fragments of the
lagging strand.
Replication of circular DNA in
E. coli (3.10):
1.
Two replication forks result in a
theta-like () structure.
2.
As strands separate, positive
supercoils form elsewhere in the
molecule.
3.
Topoisomerases relieve tensions in
the supercoils, allowing the DNA to
continue to separate.
In prokaryotes, there are three main types of DNA polymerase
Polymerase
Polymerization (5’-3’)
Exonuclease (3’-5’)
Exonuclease (5’-3’)
I
Yes
Yes
Yes
400
II
Yes
Yes
No
?
III
Yes
Yes
No
10-20
•3- 5’ exonuclease activity = ability to remove nucleotides from the
#Copies
3’ end of the chain
•Important proofreading ability
•Without proofreading error rate (mutation rate) is 1 x 10-6
•5- 3’ exonuclease activity functions in DNA replication and repair.
Eukaryotic enzymes:
Five common DNA polymerases from mammals.
1.
Polymerase  (alpha): nuclear, DNA replication, no proofreading
2.
Polymerase  (beta): nuclear, DNA repair, no proofreading
3.
Polymerase  (gamma): mitochondria, DNA repl., proofreading
4.
Polymerase  (delta): nuclear, DNA replication, proofreading
5.
Polymerase  (epsilon): nuclear, DNA repair (?), proofreading
•
•
•
•
Different polymerases for the nucleus and mtDNA
Some polymerases proofread; others do not.
Some polymerases used for replication; others for repair.
Polymerases vary by species.
DNA elongation:
Why are RNA primers used in DNA replication
•
DNA Polymerase, the main enzyme involved in DNA synthesis requires a template, and a primer
(the -OH group on the THIRD carbon (not the second) of deoxyribose). This -OH group is located on
the previous nucleotide. The phosphate group ( -PO4 ) of the incoming nucleotide will be added to
the ( -OH ) group of the previous nucleotide.
•
So now for the answer. Since the DNA polymerase needs to "see" a 3 prime hydroxyl group ( -OH )
in order to proceed adding new bases along the template, "How does synthesis start in the first
place?"
•
The answer comes from RNA polymerase (the enzyme responsible for synthesizing RNA from a
DNA template).
•
At the start of DNA replication, RNA polymerase creates a short temporary primer on the DNA
template. Like DNA polymerase, the RNA polymerase requires a template, BUT unlike DNA
polymerase, the RNA does NOT need to see that -OH group of the previous nucleotide. After a short
RNA primer sequence has been created on the DNA template strand, the RNA polymerase falls off
and DNA polymerase can add onto the hydroxyl group (-OH) of the RNA primer and start
synthesizing from there. The RNA is removed shortly after the DNA synthesis starts. RNA is used
because it is easily made for a temporary purpose such as replicating DNA and then degraded. DNA is used when the cell
wants something permanent
Replication forks visible in Drosophila
Final Step - Assembly into Nucleosomes:
•
As DNA unwinds, nucleosomes must disassemble.
•
Histones and the associated chromatin proteins must be duplicated by new
protein synthesis.
•
Newly replicated DNA is assembled into nucleosomes almost immediately.
•
Histone chaperone proteins control the assembly.
Concepts and terms to understand:
• Why are topoisomerase and helicase required?
• The difference between a template and a primer?
• The difference between primase and polymerase?
• What is a replication fork and how many are there?
• Why are single-stranded binding (SSB) proteins required?
• How does synthesis differ on leading strand and lagging strand?
• Which is continuous and discontinuous?
• What are Okazaki fragments?
THANKS