The mechanism of DNA replication

Question 1

Plausible models for DNA replication

Answer 1

Semiconservative
Conservative
Dispersive

Question 2

Semiconservative

Answer 2

Each daughter DNA contains 1 strand of parent DNA

Question 3

Conservative

Answer 3

Daughter DNA doesn’t contain any parent DNA

Question 4

Dispersive

Answer 4

Daughter DNA consist of strands each containing

segments of both parental DNA strands

Question 5

DNA replication is

Answer 5

semiconservative

Each daughter DNA contains 1 strand of parent DNA

Question 6

Meselson Stahl experiment

Answer 6

• 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

Question 7

The Mechanism of DNA Replication

Answer 7

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.

Question 8

what is the Origin of replication?

Answer 8

• 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

Question 9

Origin of replication in Prokaryotes

Answer 9

• 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

Question 10

Origin of replication in Eukaryotes

Answer 10

• 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

Question 11

The Mechanism of DNA Replication- role of protein

Answer 11

• 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

Question 12

DNA replication essential

Answer 12

– ATP, TTP, GTP, CTP (nucleotides)
– DNA polymerase
– Primer
- template

Question 13

is DNA replication bidirectional or uni- directional

Answer 13

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

Question 14

Helicase

Answer 14

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

Question 15

SSB

Answer 15

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

Question 16

The DNA replication fork

Answer 16

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.

Question 17

DNA polymerase binds to the template strand

Answer 17

• 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

Question 18

role of Primer

Answer 18

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.

Question 19

what is a primer ?

Answer 19

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.

Question 20

Sliding clamp

Answer 20

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!

Question 21

The Two New Strands Form in Different Ways

Answer 21

Leading strand: synthesising in continuous manner from 5’ prime to 3’ prime
Lagging strand: lagging strand synthesizing in discontinuous manner

Question 22

synthesizing leading strand

Answer 22

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.

Question 23

Synthesizing lagging strand

Answer 23

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.

Question 24

Step in DNA synthesis

Answer 24

1. DNA helicase and single-stranded DNA binding (SSB)
   proteins help open up the DNA helix
2. DNA primase kick-starts the DNA polymerase
3. Clamp increases the processivity of DNA polymerase
4. DNA polymerase catalyses nucleotide polymerisation

Question 25

“Tidying up”:

Answer 25

1. DNA topoisomerases to relieve helical winding
2. RNase H removes the RNA primers, which are replaced by
   DNA
3. DNA ligase seals together discontinuously synthesized
   lagging-strand DNA fragments

Question 26

Folding of lagging strand brings each completed Okazaki fragment

Answer 26

Folding of lagging strand brings each completed Okazaki fragment close to the start site for the next fragment

Question 27

High Fidelity DNA synthesis

Answer 27

Rate of roughly 1 nucleotide change per 10^9 nucleotides each time DNA is replicated