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Unit 2 B: Replication and Repair
Unit 2 B: Replication and Repair
Unit 2 B: Replication and Repair
01/04/2022
Properties of DNA Pol III:
This is the major replicating enzyme in E. coli.
E. coli polymerase III is a very complex enzyme in its most active form (pol
III holoenzyme).
The term holoenzyme refers to an enzyme that contains several different
subunits and retains some activity even when one or more subunit is missing.
The smallest aggregate having enzymatic activity is called the core
enzyme.
Genes encoding five of the subunits have been identified.
Joining linear DNA fragments together with covalent bonds is called ligation.
DNA Pol can not joint 3΄-OH and a 5΄-mono phosphate.
The joining of these groups is accomplished by the enzyme DNA ligase.
E.coli DNA ligase can join a 3΄-OH and 5΄ mono phosphate as long as both
groups are termini of adjacent base paired deoxynucleotides. i.e., the enzyme
cannot bridge a gap.
Since DNA ligase has only a monophosphate to work with, it needs another
source of energy.
It obtains energy by coupling ligation with hydrolysis of either ATP or NAD
(nicotinamide adenine dinucleotide).
The energy source depends upon the organism from which DNA ligase is
obtained.
The E. coli DNA ligase uses NAD as energy source; whereas T4 DNA ligase
needs ATP.
DNA Gyrase or Topoisomerase:
As the strands of DNA are separated at the replication fork, the dsDNA in
front of the fork becomes increasingly, positively supercoiled (compensating
winding up).
This kind of tightening of the helix will create intolerable strain unless it is
relieved.
Cairns, recognized this problem in 1963. He proposed a ‘Swivel’ in the DNA
duplex, that would allow the DNA strands on either side to rotate to relieve the
strain.
The enzyme known as DNA Gyrase serves the swivel function.
DNA gyrase belongs to a class of enzymes called topoisomerases that
introduce transient single or double stranded break into DNA and thereby
allow it to change its topology (form).
Based on the ability to cause single or double stranded breaks in DNA,
topoisomerase is classified into class I and class II.
Class I enzyme introduces temporary single strand breaks and seals.
Class II enzyme breaks and seals both strands.
Topoisomerase II is often used in E.coli replication.
These enzymes play a crucial role in replication.
Any mutation in genes of DNA gyrase is found to be lethal.
DNA Replication Models:
Bidirectional Replication:
(i) θ – replication
The first demonstration that E. coli DNA replicates as a circle came from an
auto-radiographic experiment.
Cells were grown in a medium containing [3H]-thymidine. So that all DNA
synthesized would be radioactive.
The DNA was isolated without fragmentation and placed on photographic
film.
Each 3H-decay exposed one grain in the film and after several months there
were enough grains to visualize the DNA with microscope.
A replicating circle is schematically like the Greek letter θ (theta). So, this
mode of replication is usually called θ replication.
two forks arise at a fixed starting point-the origin of replication-and move in
opposite directions around the circle until they meet on the other side.
(ii) Displacement loop or D-loop model:
At the same time, the parental template for lagging strand synthesis is
displaced.
The polymerase used for this synthesis is apparently Pol III holoenzyme.
The displaced parental strand is replicated in the usual way by means of
precursor fragments.
DNA can be damaged in many different ways, and this damage, if left
untreated, can lead to mutations: changes in the base sequences of a DNA.
Mismatch repair system must scan the genome for mismatches and
the system must correct the mismatch accurately i.e., it must replace
the misincorporated nucleotide in the newly synthesized strand and
not the correct nucleotide in the parental strand.
In E. coli, mismatches are detected by a dimer of the mismatch repair
protein MutS.