The mechanism of DNA replication Flashcards
Plausible models for DNA replication
Semiconservative
Conservative
Dispersive
Semiconservative
Each daughter DNA contains 1 strand of parent DNA
Conservative
Daughter DNA doesn’t contain any parent DNA
Dispersive
Daughter DNA consist of strands each containing
segments of both parental DNA strands
DNA replication is
semiconservative
Each daughter DNA contains 1 strand of parent DNA
Meselson Stahl experiment
• The classic Meselson and Stahl experiment proved
replication of DNA to be semiconservative.
• Each parent strand is a template for synthesis
of a new strand.
• Two replicated DNA helices contain one parent
strand and one newly synthesized strand each
The Mechanism of DNA Replication
DNA polymerase catalyzes the addition of nucleotides to
the 3’ end of the growing strand.
• Nucleotides are added by complementary base pairing
with the template strand.
• The substrates, deoxyribonucleoside triphosphates
(dNTPs), are hydrolyzed as added, providing the energy
required for DNA synthesis.
what is the Origin of replication?
- Process of DNA replication begins with special
initiator proteins that bind to double stranded DNA
to pry the two strands apart, breaking the hydrogen
bonds between the bases
• Positions where DNA helix is first opened are called
replication origins
• These sites have short sequences to attract the
initiator proteins and stretches of DNA that are easy
to open
Origin of replication in Prokaryotes
• The small circular chromosomes of prokaryotes
have a single origin of replication
Bacteria typically have a single origin of DNA
replication (500 – 1000 nt/s)
– Highly regulated
– Only initiated when sufficient nutrients
– Blocked until A’s are methylated
Origin of replication in Eukaryotes
• Large, linear DNAs have many origins.
• Eukaryote chromosomes can have hundreds of
origins of replication, and replication occurs at
many different sites simultaneously.
Eukaryotic chromosomes contain multiple origins of
replication (50 nt/s)
– Packed in chromatin
if there was a single origin of replcation , if it mutated
the DNA would not be able to replicate
The Mechanism of DNA Replication- role of protein
• Many proteins assist in DNA replication.
• Both strands of DNA act as templates.
• Localized unwinding (denaturation) of DNA
takes place at the origin of replication, via DNA
helicase.
• Single-strand binding proteins bind to the
unwound strands to keep them apart
DNA replication essential
– ATP, TTP, GTP, CTP (nucleotides)
– DNA polymerase
– Primer
- template
is DNA replication bidirectional or uni- directional
Additional proteins: increase efficiency and allow bidirectionality
• Begins at a specific point, called the origin, and
proceeds bidirectionally at the replication forks
- template
allow replication to go in both direction at the same time
Helicase
DNA helix opened up by helicase
– Enzyme that breaks hydrogen bonds holding
strands together
DNA helicase hydrolyses ATP and propel rapidly along a DNA single strand, prying apart the helix at rates up to 1000 nucleotide pairs per second
SSB
The unwound DNA is stabilized by single strand
binding protein (SSB)
SSB proteins bind tightly and cooperatively to exposed single stranded DNA without covering
the bases prevent the reuniting of the strands
The DNA replication fork
Localised region of replication
Y shaped structure – called replication fork
The point at which the two strands of DNA are separated to allow replication of each strand.
DNA polymerase binds to the template strand
• DNA polymerase is a large protein with a groove that
the DNA attaches to and can slide through.
• Cells have more than one kind of DNA polymerase.
• Shape is like a hand; the “finger” regions have
precise shapes that recognize the shapes of the
nucleotide bases
role of Primer
A DNA polymerase cannot start a strand without a primer.
• A DNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added.
what is a primer ?
A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. … The
synthesis of a primer is necessary because the enzymes that synthesize DNA, which are called DNA polymerases, can only attach new DNA nucleotides
to an existing strand of nucleotides.
Sliding clamp
A DNA clamp, also known as a sliding clamp or β-clamp, is a protein complex
that serves as a processivity-promoting factor in DNA replication. As a cr
itical component of the DNA polymerase III holoenzyme, the clamp pro
tein binds DNA polymerase and prevents this enzyme from dissociating f
rom the template DNA strand.
Clamp assembled with the clamp loader
next to the 3’ end of the growing chain
Clamp loader dissociates once clamp assembled
Onward goes the clamped polymerase!
The Two New Strands Form in Different Ways
Leading strand: synthesising in continuous manner from 5’ prime to 3’ prime
Lagging strand: lagging strand synthesizing in discontinuous manner
synthesizing leading strand
DNA polymerase action causes the leading strand to grow in
the 5’-to-3’ direction until replication of that section of DNA is complete.
RNA primer is degraded by RNase H and DNA replaces it.
Synthesizing lagging strand
On the lagging strand, growing in the other direction, DNA is
made in the 5’-to-3’ direction but synthesis is
discontinuous: DNA is added as short fragments (Okazaki
fragments) to RNA primers.
• RNase H removes the RNA primers
• DNA ligase fills in gaps between adjacent Okazaki fragments.
Step in DNA synthesis
- DNA helicase and single-stranded DNA binding (SSB)
proteins help open up the DNA helix - DNA primase kick-starts the DNA polymerase
- Clamp increases the processivity of DNA polymerase
- DNA polymerase catalyses nucleotide polymerisation
“Tidying up”:
- DNA topoisomerases to relieve helical winding
- RNase H removes the RNA primers, which are replaced by
DNA - DNA ligase seals together discontinuously synthesized
lagging-strand DNA fragments
Folding of lagging strand brings each completed Okazaki fragment
Folding of lagging strand brings each completed Okazaki fragment close to the start site for the next fragment
High Fidelity DNA synthesis
Rate of roughly 1 nucleotide change per 10^9 nucleotides each time DNA is replicated
