Ionizing radiation causes three types of damage to DNA:
Single-strand breaks - mostly sealed by DNA ligase so don't contribute
Double-strand breaks - often lethal because can't be resealed by ligase
so degraded by nucleases
Alteration of bases - this type of oxidative damage is usually lethal because
it forms a replication barrier at that site
There are four ways to repair of T dimers in E. coli:
UV radiation - 260 nm is wavelength at which maximum absorption occurs
UV - major photoproduct is intrastrand linkage of adjacent pyrimidines,
usually thymines, called thymine dimers. Creates distortion in helix and
affects replication and transcription.
Pol III can't replicate past T-dimer because if puts in A across from dimer,
recognizes the weak H-bonding as a mismatch and proofreads. Tries to put
in another A, fails. Causes stuttering of Pol III at this site.
Block in replication isn't permanent; can have dimer repaired.
(aka Light Repair)
phr gene - codes for deoxyribodipyrimidine
photolyase that, with cofactor folic acid, binds in dark to T dimer. When
light shines on cell, folic acid absorbs the light and uses the energy
to break bond of T dimer; photolyase then falls off DNA
(2) Excision Repair (aka Dark Repair)
3 different types of repair mechanisms which use different enzymes but
none-the-less follow the same basic principle as outlined in the figure
(a) AP Repair (aka Base Excision
Repair of apurinic and apyrimidinic sites on DNA in which base has
been removed. Base removed by radiation or DNA glycosylases which sense
and remove damaged bases.
ung gene codes for uracil-DNA glycosylase which recognizes and
removes U in DNA by cleaving the sugar-nitrogen bond to remove the base.
AP endonucleases: class I nick at 3' side of AP site and class II nick
at 5' side of AP site. Exonuclease removes short region of DNA and DNA
Pol I and ligase fill in gap.
(b) UV Damage Repair (also called NER -
nucleotide excision repair):
NER differs from BER in several ways.
Excinuclease (an endonuclease; also called correndonuclease [correction
endo.]) that can detect T dimer, nicks DNA strand on 5' end of dimer (composed
of subunits coded by uvrA, uvrB and uvrC genes).
It uses different enzymes.
Even though there may be only a single "bad" base to correct, its nucleotide
is removed along with many other adjacent nucleotides; that is, NER
removes a large "patch" around the damage.
UvrA protein and ATP bind to DNA at the distortion.
UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP
complex for irradiated DNA.
UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer.
UvrD (DNA helicase II; same as DnaB used during replication initiation)
separates strands to release 12-bp segment.
DNA polymerase I now fills in gap in 5'>3' direction and ligase seals.
polA - encodes DNA pol I; mutant was viable (retained normal
5'>3' exo. activity and only 2% of polymerase activity) so Pol I not major
replication enzyme, but mutant also had somewhat increased sensitivity
to UV so first suggested that played a role in DNA repair.
(c) Mismatch Repair (MMR):
Accounts for 99% of all repairs
Follows behind replication fork.
Two ways to correct mistakes made during replication:
1) 3'>5' exonuclease - proofreading
2) Mismatch repair
mutH, mutL, mutS and mutU gene products
involved (mut for mutator because if gene is mutated, cell has increased
levels of spontaneous mutations)
How does system recognize progeny strand rather than parent strand
as one with mismatch? Because of methylation. DNA methylase (coded for
by dam [DNA adenine methylase] locus) methylates 5'-GATC-3' sequence
in DNA at A residue. Mismatch from replication recognized by mutL
and mutS gene products. mutH gene product nicks DNA strand
(progeny strand) on either side of mismatch. DNA helicase II from mutU
gene (also called uvrD gene) unwinds DNA duplex and releases nicked
region. Gap filled in by DNA Pol I and ligase.
(3) Postreplicative (Recombinational)
Translesion Bypass Repair
If T dimer is not repaired, DNA Pol III can't make complementary strand during
replication. Postdimer initiation - skips over lesion and leaves large
gap (800 bases). Gap may be repaired by enzymes in recombination system
- lesion remains but get intact double helix.
RecA - coats ssDNA and causes it to invade dsDNA. When stimulated by
presence of ssDNA, it also acts as protease to cleave lambda repressor and
acts to cause autocatalysis of LexA repressor.
recA mutants - very UV-sensitive
Now have sister-strand exchange - a type of recombination
Postreplicative repair is part of SOS response.
(4) SOS Response
UV light is considered be mutagenic - why is this so if all of the UV damage
we've talked about is repaired by the 3 repair mechanisms that we have
discussed above? In all the three cases, (photoreactivation, excision repair
and postreplicative repair) are not
mutagenic as the damage is repaired.
occurs when cells are overwhelmed by UV damage - this allows the cell to
survive but at the cost of mutagenesis.
response is only triggered when other repair systems fail as they are overwhelmed
by the increased amount of damage so that unrepaired DNA accumulates in
The accumulation of DNA damage leads to repair induction or W-reactivation
Irradiated lambda phage are more likely to survive in an irradiated rather
than. an unirradiated host because SOS system has already been turned on
in irradiated host.
What triggers the SOS response? Is it the UV-irradiation event or
the presence of damaged DNA that triggers the response? If F' cell is UV-irradiated
and conjugated to unirradiated F- cell, damaged F' DNA causes induction
of repair system (SOS) with maximal enzyme levels in 30 min.
SOS regulon includes lexA (autoregulation), recA, uvrA,
LexA normally represses about 18 genes
sulA and sulB, activated by SOS system, inhibit cell
division in order to increase amount of time cell has to repair damage
Each gene has SOS box in promoter. LexA binds SOS box to repress expression.
However, LexA catalyses its own breakdown when RecA is stimulated by ssDNA.
Non-SOS repair is basically error-free, but SOS repair is error-prone.
This is why UV is a mutagen. May be due to RecA binding ssDNA in lesions,
which could then bind to DNA Pol III complex passing through this area
of the DNA and inhibit 3'>5' exonuclease (proofreading) ability. This makes
replication faster but also results in more mutations.
This affect on proofreading seems to involve UmuD'-UmuC complex as
well. RecA facilitates proteolytic cleavage of UmuD to form UmuD'. The
UmuD'-UmuC complex may bind to the RecA-Pol III complex and promote error-prone
Also allows Pol III to replicate past a T-dimer but introduces many
mutations while doing so
Once damage is repaired, RecA no longer catalyzes cleavage of LexA
(which is still being made), so uncleaved LexA accumulates and turns the
SOS system off.
Why are DNA Repair Systems Necessary?
These types of repair, capable of repairing thymine dimers, are so important
to bacteria that an E. coli strain that is phr (no photoreactivation),
(no translesion by pass or SOS), and uvrA (no excision repair) is
killed by a single thymine dimer.
Xeroderma Pigmentosum (XP):
XP is a rare inherited disease of humans which, among other things,
predisposes the patient to
pigmented lesions on areas of the skin exposed to the sun and
an elevated incidence of skin cancer.
It turns out that XP can be caused by mutations in any one of several
genes - all of which have roles to play in NER. Some of them:
XPA, which encodes a protein that binds the damaged site and helps assemble
the other proteins needed for NER.
XPB and XPD, which are part of TFIIH. Some mutations in XPB and XPD
also produce signs of premature aging. [Link]
XPF, which cuts the backbone on the 5' side of the damage
XPG, which cuts the backbone on the 3' side.
Deinococcus radiodurans, a superbug which survives radiation,
eats toxic waste:
A can of spoiled meat and nuclear waste may appear to have little
in common, but the bacterium D. radiodurans thrives in both environments.
This bacterium was discovered in 1956 when it was identified as the culprit
in a can of spoiled ground beef thought to be radiation ”sterilized.” Scientists
subsequently learned that its extreme radiation resistance enables
the microbe to survive doses thousands of times higher than would
kill most organisms, including humans. Although the ability of the
lowly cockroach to withstand radiation has long been admired, it is far
surpassed by that of the bacterium D. radiodurans. The remarkable
DNA-repair processes of D. radiodurans allow it to stitch
together flawlessly its own radiation-shattered genome in about 24 hours.
Summary for DNA transfer, recomination
||The radiation-resistance profile of D. radiodurans compared
to such other organisms as the common intestinal bacterium E.
coli, cockroaches, and humans. When older colonies of D. radiodurans
are used, their survival extends much farther, to around 17kGy (1.7 million
rads). Scientists believe this extreme radiation resistance may be a side
effect of D. radiodurans' ability to survive severe dehydration, which
also fragments DNA. [Nature Biotechnology 18, 85-90 (January 2000)]
Send comments, errors and suggestions to: B.Patel@griffith.edu.au
Created: 17 Sep 2002
Modified: 19 Sep 2002