1. DNA can be damaged through various mechanisms including base substitutions during replication, base changes due to instability, and damage from chemicals and environmental agents. 2. This document describes different types of DNA damage including mismatches, missing or altered bases, single and double strand breaks, and cross-linking. 3. DNA repair mechanisms are discussed, including mismatch repair, removal of incorrect bases by uracil glycosylase, and light-dependent and light-independent repair of thymine dimers through excision and recombinational repair.

1. Dr. N.Banu
Associate Professor
VISTAS

2. DNA REPAIR
 Alterations of DNA molecules:
1. Base substitutions during replication.
2. Base changes due to instability of the base or of the N-
glycosylic bond and
3. Due to chemicals and environmental agents.
These mechanisms are responsible for the following defects:
1. An incorrect base in one strand that cannot form
hydrogen bond with the corresponding base in the other
stand.(i) failure of editing function or (ii) spontaneous
loss of amino group, converting c to u or A to
hypoxanthine.
2. Missing bases: Depurination – loss of purine from DNA.
3. Altered bases: Bases are converted to diff. compounds by
chemical and physical agents. E.g. β-particles from
radioisotopes and X-rays. E.g. thymine dimers.

3. DNA damage
 4. single – strand breaks: A variety of agents can break
phophodiester bonds. E.g. peroidases, sulfhydryl
compounds(cysteine) and metal ions such as Fe 2+
and Cu2+. Repaired by DNA ligase.
5. Double – strand breaks: If a DNA molecules receives a
large no. of randomly located single-stranded breaks,
two breaks may be opp. One another, makes break of
the double helix.
6. Cross-linking: some antibiotic (mitomycin C) and
some reagents (nitrite ion) form covalent linkages.

4. Types of DNA Damage Summarised
G A CT
ds DNA Break Mismatch
Thymidine dimer
AP site
Covalent X-linking
ss Break
C-U deamination

5. REPAIR OF INCORRECT BASES
 Addition of incorrect base by Pol.I and III, cannot form
hydrogen bond with template. This by corrected by editing
function. This is called mismatch repair.
 Deamination: cytosine loses an amino group forming
uracil. After one round of replication lead to the
replacement of G.C pair by A.U pair, which would become
an A.T pair after another round of replication.
 1. Removal of the U by Uracil N-glycosylase. It cleaves the
N-glycosylic bond and leaves the deoxyribose in the
backbone.
 2. AP endonuclease (AP – apurinic acid) makes a single cut,
freeing one end of the deoxyribose. This is followed by
removal of deoxyribose and adjacent nuclotides
(endonuclease activity of Pol.I) after which Pol.I fills the
gap with correct nuclotides.

6. REPAIR OF THYMINE DIMERS
 1. Light – induced repair (photoreactivation)
 2. light independent repair ( dark repair)
 i. excision of the damaged base (excision repair)
 ii. Reconstruction of a functional DNA molecule from
undamaged fragment (recombinational repair) and
 Iii. Disregard of the damage (SOS repair)

7. PHOTOREACTIVATION
 It is an enzymatic cleavage of thymine dimers
activated by visible light (300-600 nm)/.
 Photoreactivation or PR enzyme is isolated from
bacteria, animals.
 It does not absorb light, not bind to any light
absorbing compound.

8. Dark repair
 Excision repair:
 It is a multistep enzymatic process.
 In E. coli – cut-patch-cut-seal.
 1. Incision step – repair endonuclease recognizes the distortion
produced by a thyamine dimer and makes a single cut in the
sugar- phosphate backbone ahead of the dimer. At the incision
site there is a 5’P group on the side of the cut containing the
dimer and a 3’OH on the other side.
 2. The 3’OH is recognized by Pol.I which synthesize new stand
while displacing a DNA segment consisting of about 20
nucleotides and carrying the thymine dimer.
 3. This segment if excised by 5’-3’ exonuclease activity of Pol.I .
 4. DNA ligase joins the newly synthesized segments.
 Incision activity is determined by 3 genes in E.coli. : uvrA,uvrB
and uvrC (ultraviolet repair endoI).
 Many human diseases may result from inability of exicision
repair. E.g. xeroderma pigmentosum.

9. 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)

10. THYMINE DIMER
 Effect of thymine dimer on Dna replication:
 Pol III reaches a thymine dimer, the rep. fork fails to
advance.
 But thymine dimer capable of forming hydrogen bond with
two adenines.
 Dimer introduces distortion into the helix.
 Editing function removes the adenine.
 The cycle begins again an adenine is added and then it is
removed. The net result is that the polymerase is stalled at
the site of the dimer. (ultraviolet light induced idling
process).

11. THYMINE DIMER
 There are 2 diff. ways in which DNA synthesis can get
going again:
 1. Post dimer initiation (Recombinational repair)
 2. Trans dimer synthesis (SOS repair)

12. RECOMBINATIONAL REPAIR
 One way to deal with a thymine dimer block is pass it by and initiate
chain growth beyond the block.
 Post dimer initiation occurs after a pause of 5 sec. per thymine dimer.
(unprimed initiation)
 The daughter strands have large gaps, because of unexcised thymine
dimers.
 There is no way to produce viable daughter cells by continued
replication alone.
 A recombination mechanism called sister-strand exchange forms
double stranded molecule.
 In sister strand exchange, a single stranded segment free of any defects
is excisied from a good strand on the homologous DNA segment at the
replication fork and inserted into the gap created by excision of a
thymine dimer.
 The combined action of Pol.I and ligase joins the inserted piece to
adjacent regions, thus filling the gap.
 The gap formed in the donor mole. By excision is also filled by pol.I
and ligase.
 Since it occurs after replication, it is called as postreplicational repair.

13. SOS REPAIR
 It is a bypass system, error-prone process, forms DNA
strands but they are defective.
 It gives relaxation to editing system and allows
polymerization to proceed across dimer (tramsdimer
synthesis) despite the distortion of the helix.
 New strands have a higher than normal no. of mispaired
bases.
 Pol. III is modified in such a way, so it can continue chain
growth without being stalled at damaged sites.
 The progeny will be mutants.
 It is cause of uv-induced mutagenesis.