Topic 7.

1: DnA ReplicAtion
DNA replication
Replication Enzymes is semi-conservative and occursReplication
during the S phase of interphase
Process

Helicase:
Helicase unwinds and separates the double stranded DNA by breaking the hydrogen
•  Helicase separates the DNA strands to form a replication fork
5’ 3’ bonds between
base pairs
(breaks the hydrogen bonds between complementary base pairs)
This occurs at specific regions (replication origins), creating a replication fork of two DNA Gyrase
•  Single stranded binding proteins prevent strands re-annealing
polynucleotide strands in antiparallel directions Helicase relieves torsion
DNA Gyrase: separates DNA
RNA
•  DNA primase synthesises
gyrase reduces a short
the torsional strainRNA
created primer on each
by helicase template strand to provide an attachment and
initiation point
•  It prevents for DNA
the DNA polymerase
from supercoiling as itIII
is being unwound
SSB
Protein
DNA Primase:
DNA polymerase III adds deoxynucleoside triphosphates (dNTPs) to the 3' end of the
•  DNA primase generates
polynucleotide a short RNA primer
chain, synthesising in aon
5' each
- 3' strand
direction
•  Primers provide an initiation point for DNA polymerase III
The(DNA pol III
dNTPs canup
pair onlyopposite
add nucleotides
their to 3’-end of a primer)base
complementary partner (adenine pairs with thymine ;
DNA Primase
guanine pairs with cytosine) makes primer
DNA Polymerase III:
•  Free nucleotides (dNTPs) line up opposite complementary bases
As the dNTPs join with the DNA chain, two phosphates are broken off, releasing the energy needed
•  DNA polymerase III covalently joins free nucleotides together
to form a phosphodiester bond DNA Pol III
Okazaki Fragments: extends chain
Synthesis
•  DNA strandsis continuous
are antiparallel,onso the strandoccurs
replication moving towards the replication fork(5’(leading
bidirectionally 3’) strand)
(replication always occurs in a 5’ 3’ direction on each strand)
Synthesis
•  Synthesis is discontinuous
is continuous on the on thestrand
leading strand moving
(towards fork) away from the replication fork (lagging strand)
leading to the formation
and is discontinuous on the of Okazaki
lagging fragments
strand (away from fork) DNA Pol I
•  Discontinuous segments are called Okazaki fragments removes primer
DNA polymerase I removes the RNA primers and replaces them with DNA
DNA Polymerase I:
•  DNA
DNA ligase joins the
pol I removes RNAOkazaki fragments
primers and together
replaces them to create
with DNA a continuousDNA
strand
Ligase
joins fragments
DNA Ligase:
DNA Gyrase reduces torsional strain created by helicase and prevents
3’ DNA from supercoiling 5’
•  DNA ligase covalently joins the Okazaki fragments together Leading Lagging

DNA Sequencing

Sequencing is a technique by which the nucleotide base order of a DNA sequence is elucidated (typically via Sanger method)
•  Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group needed to form covalent bonds (they terminate replication)
•  Four PCR mixtures are prepared – each with stocks of normal bases and one dideoxynucleotide (ddA, ddT, ddG, ddC)
•  Whenever the dideoxynucleotide is randomly incorporated, the DNA sequence is terminated at that base position
•  Because a complete PCR cycle generates millions of sequences, every base position is likely to have been terminated
•  These sequences are separated by gel electrophoresis to determine base sequence (according to ascending sequence length)
•  Automated machines can determine the sequence quickly if fluorescent labeling of the dideoxynucleotides has occurred
T A G C
T

T C
ddT ddA ddG ddC
T C G
PCR Gel Data G A C T G A AG C T
5’ 3’
C T G A C T T C G A T C G A