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.

S.n Protein Gene Role

o.
1. DNA E (α – dna E Polymerizing
subunit) activity
2. DNA Q (ε – dna Q 3→5΄ exonuclease
subunit) or edition
function
3. DNA N dna N Confer catalytic
4. DNA X dna X efficiency and
5. DNA Z dna Z high processivity
 Although Pol III shares with a Pol I a requirement for a
template and a primer, its substrate specificity is much more
limited.
 Pol III cannot act at a nick, and it is not active with single-
stranded DNA primed with RNA, without the presence of
additional protein factors.
 Pol III cannot carryout strand displacement because another
system is needed to unwind the helix.
 Although Pol III possesses 5 → 3΄ exonuclease activity, the
biological role of which is unknown.
DNA Ligase:

 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:

 DNA that contains an origin of replication form bubble shaped replication

intermediate which will become Y-shaped replication fork later on.
Since the leading strand displaces the unreplicated parental strand, the
bubble is called a displacement loop or D-loop.
 Such conformation is a transient one and it exists only until synthesis of
precursor fragment begins.
 In a replication system, that does not employ DNA gyrase to relieve
topological constrains, a D loop may be long-lived.
 In animal cell mitochondrial DNA, 70% of all replication are in the D-loop
conformation.
DNA replication in B. subtilis is bidirectional using low-radioactivity pulse
and high-radioactivity pulse.
(iii) Rolling circle replication:

 In the course of replication, a circular phage DNA molecule gives rise

to linear daughter molecules.
 The base sequence of the DNA present in the phage is repeated
numerous times in the daughter molecule forming a concatemer.
 These concatemers are usually an essential intermediate in phage
production.
 This mode of replication occurs by covalent extension, in which the
leading strand is covalently attached to a parental strand.
 This replication mode is known as rolling circle replication.
 Consider a duplex in which a nick is made having 3’-OH and 5’-P termini.
 Under the influence of a helicase and SSB protein, a replication fork can
be generated.

 Synthesis of a primer is unnecessary because of the 3’-OH group.

 Leading strand synthesis can proceed by elongation from this 3’-OH
group.

 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.

 The result of this mode of replication is a circle with a linear branch: it

resembles the Greek letter sigma (σ) and is called σ replication or rolling
circle replication.
 the rolling circle mechanism is not confined to production of single-
stranded DNA.
 some phages (λ) use this mechanism to replicate double-stranded DNA.
UNIDIRECTIONAL REPLICATION:

 A small number of phages and plasmids use the unidirectional mode

exclusively (eg. Phage col E1). A stop signal must exist that prevents
chain growth of the first precursor fragment.

 Unlike the bidirectional replication, in unidirectional replication, one

branch point remains at a fixed position with respect to the bubbles.
This position defines the replication origin.

 Completion of DNA replication requires a set of specific events.

 These events are different for circular and linear chromosomes.
 DNA Damage and Repair

 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.

 DNA damage is simply a chemical alteration to DNA.

 For example, change from G-C pair to methyl G-C; the methyl-G is likely to
mispair with T instead of C during DNA replication. If this happens, then
another round of replication will place an A across from the mispaired T,.

 A mutation is a change in a base pair (change from G-C pair to A-T).

 Two common examples of DNA damage are: base modifications caused by
alkylating agents and pyrimidine dimers caused by ultraviolet radiation.
 Alkylating agents like ethylmethane sulfonate add alkyl groups
to bases.
 Some of these alkylations do not change base-pairing, so they
are innocuous.
 Others cause DNA replication to stall, so they are cytotoxic, and
can lead to mutations if the cell attempts to replicate its DNA
without repairing the damage.
 Damage caused by ultraviolet radiation

UV radiation cross-links adjacent pyrimidines on the same

DNA strand, forming pyrimidine dimers.

 UV rays have comparatively low energy, and they cause a

moderate type of damage: pyrimidine dimers.

 Gamma and X-rays are much more energetic. They ionize

the molecules around DNA and form highly reactive free
radicals that can attack DNA, altering bases or breaking
strands.
DNA Repair:

One way to cope with DNA damage is to repair it, or restore it

to its original, undamaged state.

There are two basic ways to do this:

1.Directly undo the damage or
2.Remove the damaged section of DNA and fill it with new,
undamaged DNA.
 DNA photolyase or photoreactivating enzyme detects and binds
to the damaged DNA site (a pyrimidine dimer).
 Then the enzyme absorbs light near UV to visible, which
activates it so it can break the bonds holding the pyrimidine dimer
together.
 This restores the pyrimidines to their original independent state.
 Finally, the enzyme dissociates from the DNA and the damage is
repaired.
 Organisms (from E. coli to human) can reverse alkylation of the
O6 of guanine.
 After DNA is methylated or ethylated, an enzyme called O6-
methylguanine methyl transferase comes on the scene to repair the
damage.
 A sulfhydryl group of the enzyme accepts the methyl group from
a guanine on the DNA, thus inactivating the enzyme (suicide
enzyme)
 Mismatch Repair System:

 A mechanism exists for detecting nucleotide mismatches and

repairing them.

 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.

 The complex of MutS and the mis-match-containing DNA recruits MutL, a

second protein component of the repair system.

 MutL, in turn activates MutH, an enzyme causes an incision or nick on

one strand near the site of the mismatch.

 Nicking is followed by the action of a specific helicase and exonuclease.

 The helicase unwinds the DNA, starting from the incision and moving in
the direction of the site of the mismatch, and the exonuclease progressively
digests the displaced single strand, extending to and beyond the site of the
mismatched nucleotide.

 This action produces a single-stranded gap, which is then filled in by DNA

polymerase III and sealed with ligase.
 Parental DNA strand is hemimethylated.
 Different exonucleases are used to remove single-stranded DNA
between the nick created by MutH and the mismatch, depending on
whether MutH cuts the DNA on the 5’ or the 3’ side of the misincorporated
nucleotide.