Dr.

Kumud Sarin
Dean
 DNA Repair
• Damaged DNA must be repaired

• If the damage is passed on to subsequent generations, then we

use the evolutionary term - mutation.

• It must take place in the germ cells - the gametes - eggs and
sperm

• If damage is to somatic cells (all other cells of the body bar germ
cells) then just that one individual is affected
 Damage from where?
• Consequences of DNA replication errors
• Chemical agents acting on the DNA
• UV light imparting energy into DNA molecule
• Spontaneous changes to the DNA
 Why repair DNA?
• DNA pol does a great job, but not good enough
• Introduces errors in about 1 in 10E7 nucleotides added, which it does
not correct
• Other mechanisms exist (as we will see) to correct many of the errors
left by the replication system
• Most mistakes and damage corrected (99% -leaving just a few - only 1 in
10E9 errors are left)
• Mutations are permanent changes left in the DNA
• Repair of non-replication related damage to the DNA must also be a
priority for the cell.
• These defects also will prevent translation and duplication of the DNA
• Cell will die.
Induced mutagenesis
Can be caused by environmental agents
that damage DNA:
• UV light
• X-rays and γ-rays
• Chemical carcinogens e.g. cigarette smoke
DNA damage can lead to mutations unless it is removed by DNA
repair enzymes Unrepaired damage can have serious
consequences
IMPORTANCE OF DNA REPAIR
Hoeijmakers, 2001
 Direct DNA Damage
• Some agents damage DNA directly
• Chemicals and light
– Chemicals - alkylating agents
– Methy and ethyl groups added to DNA bases
• This type of damage can be repaired by direct reversal
involving special enzymes
– They remove the offending atoms and restore the base
 DNA Damage
• Just a few types of damage is repaired via
simple reversal of the chemical change -
– UV light induced dimers
– Methylation of bases
– Ethylation of bases
– Large chemical groups added to the DNA
• Most other damage require other systems…
 Random photons of ultraviolet (UV) light induce aberrant bonding between
06_24_radiation.jpg
neighbouring pyrimidines (thymine & cytosine) bases on the same strand of DNA. The
will prevent the replication machine from duplicating the DNA. The cell will die!

This type of defect can be readily reversed by a process called photoreactivation. Visible light
energy is used to reverse the defect (in bacteria, yeasts, protists, some plants, and some
animals but NOT in humans)
Direct Repair:
• The damage is reversed by a repair enzyme which is called
photoreactivation.
• This mechanism involves a light dependant enzyme called DNA
photolyase.
• The enzyme is present in almost all cells from bacteria to
animals.
• It uses energy from the absorbed light to cleave the C-C bond of
cyclobutyl ring of the thymine dimers. In this way thymine dimers
are monomerized.
Photoreactivation
• Catalyzed by DNA-Photolyase (DPL)
• Reverses cyclobutyl pyrimidine dimers resulting from UV
irradiation.
• It has low abundance (10 to 20 molecules per cell) in E. coli.
• DPL binds irradiated DNA 100x better than non-irradiated DNA.
• DPL contains a non-covalently associated chromophore (FADH or
FADH2).
• The photochemical mechanism of photorepair has been proposed
to be light-dependent redox reaction between the singlet excited
state of FADH2 and the pyrimidine dimer.
• Photolyase has not yet been identified in placental mammals.
Photoreactivation .
Aberrant Methylation
Catalyzed by 6-O-methylguanine methyltransferase (6-O-MGM)
6-O-MGM recognizes 6-O-methylguanine in DNA and removes the
methyl group, transferring the group to an amino acid on itself in a
"suicide" mechanism.
6-O-MGM is encoded by the ada gene in E. coli and the MGMT gene
in eukaryotes.
Mice knocked out for MGMT are cancer-prone and sensitive to
methylation agents
Photoactivation Repair in E. coli

• Exposing UV treated cells to blue light results in a reversal of the

thymine dimer formation
• Enzyme, photoactivation repair enzyme (PRE) absorbs a photon
of light (from blue light) and is able to cleave the bond forming the
thymine dimer.
• Once bond is cleaved, DNA is back to normal
Cellular Protection From DNA Damage
• Natural errors: polymerase base selection, proofreading,
mismatch repair

• Endogenous/exogenous DNA damage: base excision repair,

nucleotide excision repair, (recombination, polymerase bypass)

• Recombination and polymerase bypass do not remove damage

but remove its block to replication. Polymerase bypass is itself
often mutagenic.
 Direct Repair
• The damage is reversed by a repair enzyme which is called photo
reactivation.
• This mechanism involves a light dependant enzyme called DNA
photolyase.
• The enzyme is present in almost all cells from bacteria to
animals.
• It uses energy from the absorbed light to cleave the C-C bond of
cyclo butyl ring of the thymine dimers.
• In this way thymine dimers are monomerized.
The cell has to pick the right strand to fix or else…
The cell has a mechanism of identifying new strand synthesis by
leaving nicks that DNA. There are enzymes which scan these new
regions looking for errors
 Correction mechanisms
• Direct reversal of damage -
Photoreactivation (bacteria, yeast, some
vertebrates - not humans) Two thymines
connected together by UV light.
• Excision Repair - removal of defective DNA.
There are three distinct types
– 1) base-excision
– 2) nucleotide-excision
– 3) mismatch repair
• Common Features of Excision-Type Repair
Pathways
• Recognition: Altered DNA is recognized and
bound by a specific damage-recognition protein.
This first step recruits other components required
for the repair reaction.
• Excision: Damaged base(s), and in some cases
adjacent nucleotides, are excised from the strand
by exonucleases, resulting in a gapped DNA.
• Resynthesis: The gap is refilled by a DNA
polymerase using the complementary strand as a
template
 Base Excision Repair
• Presence of the Uracil in DNA is a great
example of this type
• Special enzymes replace just the defective
base
– 1 snip out the defective base
– 2 cut the DNA strand
– 3 Add fresh nucleotide
– 4 Ligate gap
• Base-Excision Repair (BER)

• Damaged bases are removed as free bases.

• BER primarily handles oxidative and alkylative damage.

• BER is thought to have an important role in aging.

• BER recognizes base deamination, oxidative damage, and other

minor base modifications.

• Five gene products are required for BER: glycosylase, AP

endonuclease, phosphodiesterase, DNA polymerase, and DNA
ligase.
• DNA glycosylase recognizes the damaged base and removes it,
generating an AP (apurinic, apyramidinic) site.
• AP endonuclease cleaves the phosphodiester bond, generating a
single-strand break with a 5'-terminal deoxyribophosphate moiety.
• The 5'-deoxyribophosphate is excised by action of a DNA
phosphodiesterase.
• The resulting single-nucleotide gap is repaired by DNA polymerase
β (beta).
• The resulting nick is sealed by DNA ligase.
• BER is relatively inefficient, due to the large number of peptides
needed to recognize each damage type.
 Nucleotide Excision
• Same as previous except that
– It recognizes more varieties of damage
– Remove larger segments of DNA (10 -100s of
bases)
• Nucleotide-Excision Repair (NER)

• Damaged bases are removed as oligonucleotides.

• NER is primarily responsible for removal of UV-induced
damage and bulky adducts, but also removes ~20% of
oxidative damage.
• Deficiencies cause many human disorders.
• Xeroderma Pigmentosum (both classical and variant)
and Cockayne's syndrome are caused by defects in
NER, resulting in various detrimental effects upon
exposure to UV light.
 Mismatch Repair
• Special enzymes scan the DNA for bulky
alterations in the DNA double helix
• These are normally caused by mismatched
bases
• AG
• AC
• CT
• These are excised and the DNA repaired
 06_26_three steps.jpg
Basic mechanism is the same
for all three types
1) Remove damaged
region
2) Resynthesis DNA
3) Ligate
Excision Repair

• Conserved throughout evolution, found in

all prokaryotic and eukaryotic organisms

• Three step process:

– 1. Error is recognized and enzymatically clipped out
by a nuclease that cleaves the phosphodiester bonds
(uvr gene products operate at this step)

– 2. DNA Polymerase I fills in the gap by inserting the

appropriate nucleotides

– 3. DNA Ligase seals the gap

Excision Repair

• Two know types of excision repair

– Base excision repair (BER)

• corrects damage to nitrogenous bases created by
the spontaneous hydrolysis of DNA bases as well
as the hydrolysis of DNA bases caused by agents
that chemically alter them

– Nucleotide excision repair (NER)

• Repairs “bulky” lesions in DNA that alter or distort
the regular DNA double helix
• Group of genes (uvr) involved in recognizing and
clipping out the lesions in the DNA
• Repair is completed by DNA pol I and DNA ligase
Proofreading and Mismatch Repair

• In bacterial systems, proofreading decreases the

error rate in DNA replication by two orders of
magnitude
– from 1 mismatch in every 105 nucleotide pairs to 1 in every
107 base pairs

• Mismatch repair is another mechanism by which

mismatches can be fixed in the DNA strand

• In bacteria, mismatch repair is based on the

process of DNA Methylation, which labels one
strand, providing a basis for the mismatch repair.
Post-Replication Repair

• Post-replication repair– Discovered in E.

coli by Miroslav Radman

– Responds when damaged DNA escapes

repair and the damage disrupts replication
– Rec A protein stimulates recombination
between donor strand and new strand
– Creates gap in donor strand which can be
repaired
– DNA Polymerase and DNA Ligase involved
 Diseases in which DNA repair is damage

• Xeroderma pigmentosum (XP): Patients are

hypersensitive to UV light; patients often develop
malignancies of the skin.

• Ataxia telangiectasia (AT): Patients are sensitive

to gamma irradiation; patients develop
neurological and skin lesions.
• Fanconi’s anemia: Patients demonstrate aplastic
anemia, growth retardation, and congenital
anomalies; related to a deficiency in repair of
DNA cross-links.
Xeroderma Pigmentosum (XP) and DNA Repair
Defects

• XP is an autosomal recessive disease associated

with dry skin, freckling, corneal ulceration, and skin tumors

• Many patients die before age 30 from metastases

of malignant skin tumors

• One form of XP is produced by a defect in the

human endonuclease that removes pyrimidine
dimers

• Mutations in at least seven other genes involved

in repairing UV-damaged DNA can cause XP
DNA Repair and Clinical Syndromes:
Increased Sensitivity;
Chromosomal Instability
and Increased Cancer Risk
 Fanconi anemia (FA)

• Fanconi anemia (FA) is an autosomal recessive

disease characterized by progressive bone marrow
failure due to defective stem cell function .
• FA cells are hypersensitive to DNA cross - linking
agents such as mitomycin C (MMC) resulting in
cytogenetic aberrations, G2 - M cell cycle arrest,
apoptosis, and cell death.
• Seven complementation groups (termed FANCA - G)
are identified. Group A (FANCA) mutations
are the most prevalent (70%).

• There are at least seven FA genes: A, C, D2, E, F, G and BRCA2

• The function of the FANC genes are still unclear.

Involvement in DNA repair system is suggested.
 Fanconi anemia (FA)

• Fanconi anemia (FA) is an autosomal recessive

disease characterized by progressive bone marrow
failure due to defective stem cell function .
• FA cells are hypersensitive to DNA cross - linking
agents such as mitomycin C (MMC) resulting in
cytogenetic aberrations, G2 - M cell cycle arrest,
apoptosis, and cell death.
• Seven complementation groups (termed FANCA - G)
are identified. Group A (FANCA) mutations
are the most prevalent (70%).

• There are at least seven FA genes: A, C, D2, E, F, G and BRCA2

• The function of the FANC genes are still unclear.

Involvement in DNA repair system is suggested.
• 2. Excision Repair:
• It includes base excision repair and nucleotide excision repair. Base excision
repair system involves an enzyme called N-glycosylase which recognizes the
abnormal base and hydrolyes glycosidic bond between it and sugar.

• Another enzyme, an endonuclease cleaves the DNA backbone on the 5′-side of

the abnormal base. Then the DNA polymerase by its exonuclease activity
removes the abnormal base. DNA polymerase then replaces it with normal base
and DNA ligase seals the region.

• Nucleotide repair system includes three steps, incision, excision and synthesis.
Incision is done by endonuclease enzyme precisely on either side of the
damaged patch of the strand. In this way damaged portion of the strand is
cleaved.
• Endonuclease enzymes involved are UvrA, UvrB which
recognize the damaged stretch of the strand. UvrC makes
two cuts (incision) on either side. Exonuclease removes the
damaged strand. Enzyme involved is UvrD.
• Later, DNA polymerase synthesizes the new strand by using
complementary strand as a template. DNA ligase forms
phosphodiester bonds which seal the ends on newly
synthesized strand.
• Mismatch Base Repair:
• Sometimes wrong bases are incorporated during
replication process, G-T or C-A pairs are formed.
The wrong base is always incorporated in the
daughter strand only.
• Therefore in order to distinguish the two strands
for the purpose of repair, the adenine bases of
the template strand are labelled or tagged by
methyl groups. In this way the newly replication
DNA helix is hemimethylated. The excision of
wrong bases occur in the non-methylated or
daughter strand.
Cellular protection from DNA damage
• Natural errors: polymerase base selection,
proofreading, mismatch repair
• Endogenous/exogenous DNA damage: base excision
repair, nucleotide excision repair, (recombination,
polymerase bypass)
• Recombination and polymerase bypass do not
remove damage but remove its block to replication.
Polymerase bypass is itself often mutagenic.
Common features of DNA polymerases
• Right hand: “palm”, “fingers”, “thumb”
• Palm --> phoshoryl transfer
• Fingers --> template and incoming nucleoside
triphosphate
• Thumb --> DNA positioning, processivity and
translocation
• Some polymerase have associated 3’ to 5’
exonuclease “proofreading” activity in a second
domain
Structures of 4 polymerase classes
•Fidelity is increased by
action of 3’ to 5’
exonuclease
“proofreading” activity
•Active site of exo is 30
Å from pol, below palm
 Contribution of proofreading, base
excision repair and MMR to mutation
avoidance
Genotype Rifr mutants per 108 cells
Wild-type mut+ 5-10
mutD (dnaQ) 4000-5000
Pol III proofreading
mutS 760
MMR
mutY mutM 8200
8-oxoG BER
 Base excision repair (BER)
• Major pathway for repair of modified bases, uracil
misincorporation, oxidative damage
• Various DNA glycosylases recognize lesion and
remove base at glycosidic bond, thereby
producing an “abasic” or AP (apurinic/
apyrimidinic) site by base “flipping out”
• One of several AP endonucleases incises
phosphodiesterase backbone adjacent to AP site
• AP nucleotide removed by exonuclease/dRPase
and patch refilled by DNA synthesis and ligation
• This repair involve the direct removal of the damaged base from
DNA .
• This serves as the trigger to activate the enzymes that excise &
replace a stretch of DNA , including the damaged site .
• Enzymes that removes bases from DNA are called glycosylases.
• DNA glycosylases are responsible for initial recognition of the
lesion.
• They flip the damaged base out of the double helix and cleave
the N-glycosidic bond of the damaged base,leaving an AP site.
• There are two categories of glycosylases:
• Monofunctional And Bifunctional.
• A wide variety of glycosylases have evolved to recognize
different damaged base.
• The AP endonucleases cleave an AP site to yield a 3' hydroxyl
adjacent to a 5' deoxyribose phosphate (dRP).
• AP endonucleases are divided into two families based on their
homology to bacterial AP endonuclease IV and exonuclease III.
Mechanism of BER
 NH2 O
4 CH3
3 HN
N 5
O 2
O
1 6
O N O N
5’ H2C H2C
O O glycosidic bond
4’ 1’

3’ 2’
O O

deoxycytosine deoxyuracil thymine

 Types of lesions repaired by BER
• Oxidative lesions; 8-oxo-G, highly mutagenic,
mispairs with A, producing GC --> TA transversions
example MutY, MutM=Fpg from E. coli
• Deoxyuracil: from misincorporation of dU or
deamination of dC-->dU, example Ung, uracil N-
glycosylase
• Various alkylation products e. g. 3-meA
• These lesions are not distorting and do not block DNA
polymerases
• Spontaneous depurination (esp. G) yield abasic sites
that are repaired by second half of BER pathway
 Mismatch repair (MMR)
• Despite extraordinary fidelity of DNA synthesis, errors do persist
• Such errors can be detected and repaired by the post-
replication mismatch repair system
• Prokaryotes and eukaryotes use a similar mechanism with
common structural features
• Defects in MMR elevate spontaneous mutation rates 10-1000x
• Defects in MMR underlie human predisposition to colon and
other cancers (“HNPCC”)
• MMR also processes mispairs that result from heteroduplex
DNA formed during genetic recombination: act to exclude
“homeologous” recombination
 Mechanism of MMR
CH3 CH3
5' 3'
3' 5'

MutS MutL MutH Initiation MutS MutL MutH

CH3 CH3 CH3 CH3
5' 3' 5' 3'
3' 5' 3' 5'

UvrD + ExoI or ExoX or ExoVII Excision UvrD + RecJ or ExoVII

CH3 CH3 CH3 CH3
5' 3' 5' 3'
3' 5' 3' 5'

PolIII + ligase Resynthesis

PolIII + ligase
CH3 CH3 CH3 CH3
5' 3' 5' 3'
3' 5' 3' 5'
 Mechanism of MMR
CH3 CH3
5' 3'
3' 5'

MutS MutL MutH Initiation MutS MutL MutH

CH3 CH3 CH3 CH3
5' 3' 5' 3'
3' 5' 3' 5'

UvrD + ExoI or ExoX or ExoVII Excision UvrD + RecJ or ExoVII

CH3 CH3 CH3 CH3
5' 3' 5' 3'
3' 5' 3' 5'

PolIII + ligase Resynthesis

PolIII + ligase
CH3 CH3 CH3 CH3
5' 3' 5' 3'
3' 5' 3' 5'
 Basis of MMR recognition
• MutS dimer (in yeast, Msh2/Msh3 or Msh2/Msh6
heterodimer)
• By DNA binding expts in vitro and DNA
heteroduplex repair expts in vivo: MMR can
recognize all base substitutions except C:C and
short frameshift loops <4 bp
• Transition mispairs G:T and A:C and one base
loops are particularly well-recognized (these are
also the most common polymerase errors)
Structure of MutS bound to DNA

60° kink in DNA

Widens minor groove,
narrows major groove
 The problem of strand discrimination
• MMR can only aid replication fidelity if repair is targeted to newly
synthesized strand
• In E. coli, this is accomplished by the transient lack of methylation of
adenines in GA*TC motifs (by the “Dam” methylase)
• MutH endonuclease cleaves only unmethylated GATC sites, allowing
entry on newly synthesized strand
• dam mutants are “mutators” and show random repair of either DNA
strand
• In other bacteria and in eukaryotes, the basis of strand discrimination
is not understood, although entry at nicks in discontinuously
synthesized DNA has been proposed
 A G
5’ 5’
5’ 5’
T C
Heat denature

A
5’
G 5’
5’ C
5’
T
Cool renature

A G
5’ 5’
5’ 5’
T C
homoduplexes
+
A G
5’ 5’
5’ 5’
C T
heteroduplexes
 In bacteriophage lambda (40 kb):
A G
5’ 5’
5’ 5’
T C
Heat denature

“light strand”

CsCl gradients
“heavy strand”

Single heteroduplex
G
5’
5’
T
G A
5’ 5’
Transfect, repair
5’ 5’
C T
 Grow in Dam+: Grow in Dam-:

A G
5’ * * * 5’
5’ 5’
* T * * C
Heat denature

“light strand”

CsCl gradients
“heavy strand”

hemi-methylated heteroduplex

G
5’
5’
* T * *
Transfect,
Methyl-directed repair
A
5’
5’
*T * *
 Comparison of eukaryotic vs.
prokaryotic MMR
• Various Msh and Mlh (Pms1) heterodimers vs.
MutS and MutL homodimers
Msh2/6 specialized for base substitution
mispairs; Msh2/3 for loop mispairs
• No MutH, Dam; basis for strand discrimination
unknown
• Basis of excision (comparable to UvrD and
Exos) incompletely understood
• Recombination Repair or Retrieval System:
• In thymine dimer or other type of damage, DNA replication
cannot proceed properly. A gap opposite to thymine dimer is left
in the newly synthesized daughter strand. The gap is repaired by
recombination mechanism or retrieval mechanism called also
sister strand exchange.
•
• DNA polymerase
• • Pol β is the main human polymerase that
catalyzes short-patch BER, with pol λ able to
compensate in its absence.
• • In addition to polymerase activity, these
enzymes have a lyase domain that removes the 5'
dRP left behind by AP endonuclease cleavage.
• • These polymerases perform displacing
synthesis, meaning that the downstream 5' DNA
end is "displaced" to form a flap
• Flap endonuclease
• • FEN1 removes the 5' flap generated during long
patch BER.
• This endonuclease shows a strong preference for
a long 5' flap adjacent to not 3' flap. In addition
to its role in long-patch BER,
• FEN1 cleaves flaps with a similar structure during
Okazaki fragment processing, an important step
in lagging strand DNA replication.
• DNA ligase
• DNA ligase III catalyzes the nick-sealing step in
short-patch BER in humans. DNA ligase I ligates
the break in long-patch BER.
• In humans, polynucleotide kinase-phosphatase
(PNKP) promotes formation of these ends during
BER.
• This protein has a kinase domain, which
phosphorylates 5' hydroxyl ends, and a
phosphatase domain, which removes phosphates
from 3'ends.
• Together, these activities ready single-strand
breaks with damaged termini for ligation.
• The base excision repair (BER) process removes
base damage such as oxidation, alkylation or abasic
sites.
• Two BER sub-pathways have been characterized
using in vitro methods, and have been classified
according to the length of the repair patch as either
'short-patch' BER (one nucleotide) or 'long-patch'
BER (LP-BER; more than one nucleotide).
• To investigate the occurrence of LP-BER in vivo, we
developed an assay using a plasmid containing a
single modified base in the transcribed strand of the
enhanced green fluorescent protein (EGFP) gene and
a stop codon, based on a single-nucleotide
mismatch, at varying distances on the 3′ side of the
lesion.
• The reversion of the stop codon occurs after
DNA repair synthesis and restores EGFP
expression after transfection of mismatch-
repair-deficient cells.
• Repair patches longer than one nucleotide
were observed for 55–80% or 80–100% of the
plasmids with a mean length of 2–6 or 6–12
nucleotides for 8-oxo-7,8-dihydroguanine or a
synthetic abasic site, respectively.
• These data show the existence of LP-BER in
vivo, and emphasize the effect of the type of
BER substrate lesion on both the yield and the
extent of the LP-BER sub-pathway.
 Nucleotide excision repair (NER)
• Recognizes bulky lesions that block DNA replication (i.
e. lesions produced by carcinogens)--example, UV
pyrimidine photodimers
• Common distortion in helix
• Incision on both sides of lesion
• Short patch of DNA excised, repaired by re-
polymerization and ligation
• In E. coli, mediated by UvrABCD
• Many more proteins involved in eukaryotes
• Can be coupled to transcription (TCR, “transcription
coupled repair”)
• Defects in NER underlie Xeroderma pigmentosum
 Xeroderma pigmentosum

•Autosomal recessive mutations in several complementation

groups
•Extreme sensitivity to sunlight
•Predisposition to skin cancer (mean age of skin cancer = 8 yrs vs.
60 for normal population)
Recognition and binding Incision Excision and repair

Nicks delivered 3’ and 5’

UvrA acts as classical “molecular to lesion by UvrBC Short fragment
matchmaker” released by helicase
action
Proteins Required for Eukaryotic Nucleotide Excision Repair

S. cerevisiae protein Human protein Probable function

Rad14 XPA Binds damaged DNA after XPC or RNA pol II
Rpa1,2,3 RPAp70,p32,p14 Stabilizes open complex (with Rad14/XPA); positions
nucleases
Rad4 XPC Works with hHR23B; binds damaged DNA;
recruits other NER proteins
Rad23 hHR23B Cooperates with XPC (see above); contains ubiquitin
domain; interacts with proteasome and XPC
Ssl2 (Rad25) XPB 3' to 5' helicase
Tfb1 p62 ?
Tfb2 p52 ?
Ssl1 p44 DNA binding?
Tfb4 p34 DNA binding?
Rad3 XPD 5' to 3' helicase
Tfb3/Rig2 MAT1 CDK assembly factor
Kin28 Cdk7 CDK; C-terminal domain kinase; CAK
Ccl1 CycH Cyclin
Rad2 XPG Endonuclease (3' incision); stabilizes full open
complex
Rad1 XPF Part of endonuclease (5' incision)
Rad10 ERCC1 Part of endonuclease (5' incision)
 Human NER

Rad1/10 Rad2 in S. cerevisiae

 Lesion bypass polymerization
• Replication-blocking lesions such as UV photodimers
can be repaired by NER but pose a serious problem if
they are in ssDNA
• As a last resort, cells employ “bypass” polymerases
with loosened specificity
• In E. coli: DinB (PolIV) and UmuD’C (Pol V); homologs
in eukaryotes; mutated in XPV
• These polymerases are “error-prone” and are
responsible for UV-induced mutation
• Expression and function highly regulated: dependent
on DNA damage
 Characteristics of lesion bypass
polymerases
• Error rate 100-10,000 x higher on undamaged
templates
• Lack 3’ to 5’ proofreading exonuclease activity
• Exhibit distributive rather than processive
polymerization (nt. incorporated per binding
event)
• Support translesion DNA synthesis in vitro
Table 1. Low-fidelity copying of undamaged DNA by specialized
DNA polymerases from human cells. [Adapted from P. J. Gearhart
and R. D. Wood, Nature Rev. Immunol. 1, 187 (2001)]
------------------------------------------------------------------------
DNA polymerase Gene Infidelity on undamaged DNA templates (relative
to pol e = ~1)
------------------------------------------------------------------------
b POLB ~50
z REV3L ~70
k POLK ~580
h POLH ~2,000
i POLI ~20,000
l POLL ?
µ POLM ?
q POLQ ?
Rev1 REV1L ?
• During replication of DNA two identical copies are produced.
Replicating DNA molecule has four strands A, B, C and D. Strands
A and C have same DNA , sequence. Strands B and D also have
same sequence as they are identical. A thymine dimer is present
in strand A. The replication fork passes the dimmer as it cannot
form hydrogen bonds with incoming adenine bases, thus creating
a gap in the newly synthesized strand B.

• In recombination repair system a short identical segment of DNA

is retrieved from strand D and is inserted into the gap of strand B.
But this creates a gap in strand D which is easily filled up by DNA
polymerase using normal strand C as a template.
• This event is dependent on the activity of a special protein
Rec A. The Rec A protein plays its role in retrieving a portion
of the complementary strand from other side of the
replication fork to fill the gap. Rec A is a strand exchange
protein.

• After filling both gaps, thymine is monomerised. So in this

repair mechanism a portion of DNA strand is retrieved from
the normal homologous DNA segment. This is also known
as daughter strand gap repair mechanism.
• SOS Repair Mechanism:
• Sometimes the replicating machinery is unable to
repair the damaged portion and bypasses the
damaged site. This is known as translesion
synthesis also called bypass system and is
emergency repair system.
• This mechanism is catalyzed by a special class of
DNA polymerases called Y-family of DNA
polymerases which synthesized DNA directly across
the damaged portion.
• The SOS response is a global response to DNA
damage in which the cell cycle is arrested and DNA
repair and mutagenesis are induced. The system
involves the RecA protein (Rad51 in eukaryotes). ...
It is an error-prone repair system that is attributed
to mutagenesis.
 SOS repair:
• 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 cell.

• The accumulation of DNA damage leads to repair induction or W-

reactivation (Weigle-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.
• "Save Our Ship". Accumulating damage to
DNA, e.g. from high doses of radiation that
break the DNA backbone, will generate single-
stranded regions in DNA.
• The increasing amounts of single-stranded
DNA induce SOS functions, which stimulate
both the recombination repair and the
translesional synthesis
• SOS REPAIR / ERROR–PRONE REPAIR :
• The SOS response is a global response to DNA
damage in which the cell cycle is arrested and DNA
repair ,mutagenesis are induced.
• The system involves the Rec-A protein (Rad51 in
eukaryotes).
• The RecA protein, stimulated by single-stranded
DNA, is involved in the inactivation of the Lex-A
repressor thereby inducing the response.
• It is an error-prone repair system that is attributed
to mutagenesis.
• The SOS response was discovered and named by
Miroslav Radman in 1975.
• The damaged DNA cause RecA to trigger the
response & results in the auto cleavage of
protein called LexA protein .
• RecA is activated on binding on a single-
stranded DNA.
• Lex A is a repressor that participate in DNA
repair . RecA forms a filament around these
ssDNA regions in an ATP-dependent fashion,
and becomes activated.
• The activated form of RecA interacts with the
LexA repressor to facilitate the LexA repressor's
self-cleavage from the operator.
• Once the pool of LexA decreases, repression of
the SOS genes goes down according to the level
of LexA affinity for the SOS boxes.
• Operators that bind LexA weakly are the first to
be fully expressed.
• In this way LexA can sequentially activate
different mechanisms of repair.
• Genes having a weak SOS box (such as uvrA,
uvrB, and uvrD) are fully induced in response to
even weak SOS-inducing treatments.
• Thus the first SOS repair mechanism to be
induced is nucleotide excision repair(NER),
whose aim is to fix DNA damage without
commitment to a full-fledged SOS response.
• This causes filamentation, and the induction
of UmuDC-dependent mutagenic repair
• Photo reactivation/direct repair :
• Photolyases are DNA repair enzymes that repair damage
caused by
• exposure to ultraviolet light. This enzyme mechanism
requires visible light(300-600 nm), preferentially from the
violet/blue end of the spectrum, and is known as photo
reactivation.
• Photolyase is a phylogenetically old enzyme which is
present and
• functional in many species, from the bacteria to the fungi
to plants and to the animals.
• Photolyase is particularly important in repairing UV induced
damage in plants.
• The photolyase mechanism is no longer working in humans
and other placental mammals who instead rely on the less
efficient nucleotide excision repair mechanism
• Photolyases bind complementary DNA strands
and break certain types of pyrimidine dimers that
arise when a pair of thymine or cytosine bases on
the same strand of DNA become covalently
linked.
• These dimers result in a 'bulge' of the DNA
structure, referred to as a lesion.
• Photolyases have a high affinity for these lesions
and reversibly bind and convert them back to the
original bases.
• The structural features that enable replicative
DNA polymerases to synthesize DNA rapidly
and accurately also limit their ability to copy
damaged DNA. Direct replication of DNA
damage is termed translesion synthesis (TLS),
a mechanism conserved from bacteria to
mammals and executed by an array of
specialized DNA polymerases.
• translesion polymerases replicate damaged
DNA and how they are regulated to balance
their ability to replicate DNA lesions with the
risk of undesirable mutagenesis
• DNA Repair Pathways
• Direct Reversal
• The simplest of the human DNA repair Pathways most
energy efficient method involves the direct reversal of
the highly mutagenic alkylation lesion O6-
methylguanine (O6-mG)
• Carried out by the product of the MGMT gene O6-
alkylguanine DNA alkyltransferase (AGT) (O6-
methylguanine DNA methyltransferase)
• Correction of the lesion occurs by direct transfer
of the alkyl group on guanine to a cysteine
residue in the active site of MGMT in a "suicide"
reaction.
• The inactivated alkyl-MGMT protein is then
degraded in an ATP-dependent ubiquitin
proteolytic pathway.
• The O6-mG adduct is generated in low levels by
the reaction of cellular catabolites with the
guanine residues in the DNA.
• A number of DNA-damaging chemotherapeutic
agents attack the O6 position on guanine, forming
the most potent cytotoxic DNA adducts known
• AGT activity correlates inversely with sensitivity
to agents that form such O6-alkylguanine DNA
adducts
• BER has two subpathways:
• – short patch: replaces the lesion with a single nucleotide

• – long patch: replaces the lesion with approximately 2 to 10

nucleotides

• Both initiated by the action of a DNA glycosylase that cleaves the N-

glycosidic bond between the damaged base and the sugar phosphate
backbone of the DNA.

• A base-specific DNA glycosylase detects an altered base and removes

it

• AP endonuclease and phosphodiesterase remove sugar phosphate.

• DNA Polymerase fills and DNA ligase seals the nick

• Recombinational Repair
• This type of repair is much more complicated than is excision
repair, and requires many more gene products.
• The products of a number of these repair genes are induced by
radiation damage, and therefore this type of repair requires
protein synthesis before it can function.
• Because of its complexity, this type of repair makes mistakes
• Postreplication Repair (recombinational DNA repair)
• (a) The dots indicate lesions in the DNA.
• (b) DNA synthesis proceeds up to a lesion and then skips past the
lesion,leaving a gap in the daughter strand.
• (c) Filling of the daughter strand gaps with DNA from parental
strands by a recombinational process that requires a functional
recA gene.
• (d) Gaps in the parental strands are repaired by repair replication
• Recombinational Excision Repair
• This process occurs in the part of the chromosome that was
replicated prior to irradiation,i .e. , where two sister duplexes
were present before irradiation.

• After the excision of the lesion, the resulting gap is f i l led by the
same recombinational process
• Multiple DNA repair pathways
• Base excision repair (BER)
• Nucleotide excision repair (NER)
• Mismatch repair (MR)
• DNA strand cross link repair
• Homologous recombination (HR)
• Non-homologous end joining (NHEJ)
• transcription coupled repair
 Spontaneous DNA alteration
• Nucleotides known to be modified by:
• Oxidative damage
• hydrolytic attack
• uncontrolled methylation
• Purines (guanine and adenine) are more affected by
those spontaneous reactions.
• 5000 purine bases are lost every day: DEPURINATION
• Spontaneous DEAMINATION of cytosine uracil occurs
at a rate of 100 bases per cell per day.
• UV can covalently link two adjacent pyrimidine bases
to form THYMINE DIMERS.
 “Risky Business”
• backup’ polymerases not as accurate as the
normal
• replicative polymerase lack exonucleotic
proofreading activity
• Restrained to telomerase in eukaryotes, using a
RNA template for DNA synthesis
• 1. Replicative polymerases
• 2. Translesion DNA synthesis (TLS) polymerases
• 3. DNA repair and recombination
• 4. Reverse transcriptase
DNA repair defects cause disease