Dr Asifa Ashraf

 DNA constantly being subjected to environmental

insults that cause the alteration or removal of
nucleotide bases.
 The damaging agents can be either chemicals , for
example, nitrous acid or radiation for example
ultraviolet light which can fuse two pyrimidines
adjacent to each other in the DNA, and high energy
ionizing radiation , which can cause double strand
breaks.
 Bases are also altered or lost spontaneously from
mammalian DNA at a rate of many thousands per cell
per day.
 If the damage is not repaired a permanent mutation
may be introduced that can result in any of a number
of deleterious effects, including loss of control over the
proliferation of the mutated cell leading to cancer.
 Cells are remarkably efficient at repairing damage
done to their DNA.
 Most of the repair systems involve recognition of the
damage on the DNA, removal or excision of the
damage, replacement or filling the gap left by excision
using the sister strand as a template for DNA synthesis
and ligation.
 These repair systems thus perform EXCISION REPAIR.
 Excision repair can be divided into NUCLEOTIDE
excision and BASE EXCISION depending on how
much of the DNA is removed.
A. Methyl directed mismatch
repair(nucleotide excision repair)
 Sometimes replication errors escape the proofreading
function during DNA synthesis, causing a mismatch of
one to several bases.
 In E-Coli mismatch repair is mediated by a group of
proteins known as Mut proteins.
 Analog proteins are present in humans.
1.Identification of mismatched
strand
 When a mismatch occurs, the Mut proteins that identify
the mispaired nucleotides must be able to discriminate
between the correct strand and the strand with the
mismatch.
 Discrimination is based on the degree of methylation.
 GATC sequences which are found approximately once every
thousand nucleotides, are methylated on the adenine
residue.
 This methylation is not done immediately after synthesis so
the newly synthesized DNA is hemimethylated( that is the
parental strand is methylated but the daughter strand is
not)
 The methylated parental strand is assumed to be
correct, and it is the daughter strand that gets
repaired.
2.Repair of damaged DNA
 When the strand containing the mismatch is
identified, an endonuclease nick the strand and the
mismatched nucleotide(s) is/are removed by an
EXONUCLEASE.
 Additional nucleotides at the 5´and 3´ends of the
mismatch are also removed.
 The gap left by removal of the nucleotides is filled , using
the sister strand as a template by a DNA polymerase.
 The 3´-hydroxyl of the newly synthesized DNA is joined
to the 5´-phosphate of the remaining stretch of the
original DNA strand by DNA ligase.
 Mutation to the proteins involved in mismatch repair
in humans is associated with HEREDITARY NON
POLYPOSIS COLORECTAL CANCER(HNPCC) ,also
known as LYNCH SYNDROME.
 With HNPCC there is an increased risk for developing
COLON CANCER, but only about five percent of all
colon cancer is the result of mutation in mismatch
pair.
B. Repair of damage caused by UV
light
 Exposure of a cell to UV light can result in the covalent
joining of two adjacent pyrimidines, producing a
DIMER.
 These thymine dimers prevent DNA polymerase from
replicating the DNA strand beyond the site of dimer
formation.
1.Recognition and excision of
dimers by UV specific
endonuclease
 First a UV specific endonuclease recognizes the dimer,
and claves the damaged strand on both the 5´ side and
the 3´side of the dimer.
 A short oligonucleotide containing the dimer is released
leaving a gap in the DNA strand that formely contained
the DIMER.
2.UV radiation and Cancer
 Pyrimidine dimers can be formed in the skin cells of
humans exposed to ultraviolet sunlight.
 In the rare genetic disease XERODERMA
PIGMENTOSUM, the cells can not repair the damaged
DNA, resulting in extensive accumulation of
mutations and consequently skin cancers.
 Xeroderma pigmentosum can be caused by defects in
any of the several genes required for UV damage
repair.
C. Correction of base
alterations(base excision repair)
 The bases of DNA can be
 1. altered either spontaneously as is the case with
cytosine which slowly undergoes deamination to form
URACIL.
 2.By the action of deaminating or alkylating
compounds . For example NITROUS ACID which is
formed by the cell from precursors such as
NITROSAMINES , NITRITES and NITRATES is a
potent compound that deaminates cytosine, adenine
and guanine.
 3.Bases can also be lost spontaneously.
 For example, approximately 10,000 purine bases are
lost this way per cell per day.
 Lesions involving base alterations or loss can be
corrected by BASE EXCISION REPAIR.
1.Removal of abnormal bases
 Abnormal bases such as URACIL, which can occur in
DNA either by deamination of cytosine or improper
use of dUTP instead of dTTP during DNA synthesis,
are recognized by specific GLYCOSYLASES that
hydrolytically cleave them from the deoxyribose back
bone of the strand .This leaves a pyrimidinic site (or a
purinic, if a purine was removed) both referred as AP
sites.
2.Recognition and repair of an AP
site
 Specific AP endonucleases recognizes that a base is
missing and initiate the process of excision and gap
filling by making an ENDONUCLEOLYTIC CUT just
to the 5´side of the AP site.
 A deoxyribose phosphate lyase removes the single ,
empty, sugar phosphate residue.
 A DNA polymerase and DNA ligase complete the
repair process.
D. Repair of double stranded
breaks
 High energy radiation or oxidative free radicals can
cause double-strand breaks in DNA, which are
potentially lethal to the cell.
 Double strand breaks also occur naturally during gene
rearrangements . dsDNA breaks cannot be corrected
by the previously described strategy of excising the
damage on one strand and using the remaining strand
as a template for replacing the missing nucleotide(s).
 Instead double strand breaks are repaired by one of
the two systems:
 1. Non homologous end joining repair, in which the
ends of two DNA fragments are brought together by a
group of proteins that affect their religation.
 This system does not require that the two DNA
sequences have any sequence homlogy .
 However this mechanism of repair is ERROR PRONE
and mutagenic.
 Defects in this repair system are associated with a
predisposition to cancer and immunodeficiency
syndrome.
 2.HOMOLOGUS RECOMBINATION REPAIR: uses the
enzymes that normally perform GENETIC
RECOMBINATION between the homologous
chromosomes during meiosis.
 This system is much less ERROR PRONE than non
homologous end joining.
Diseases of DNA Repair mechanism
 1 .XERODERMA PIGMENTOSUM: It is an autosomal
recessive condition. Defect lies in the NER(nucleotide
excision repair) mechanism; any of the seven genes
involved in this mechanism may be defective.
 There is the sensitivity to ultraviolet rays; sunlight
causes blisters on the skin.
 Death usually occurs in the second decade of life due
to squamous cell carcinoma of skin.
 2.ATAXIA TELANGECTASIA : It is a common
autosomal recessive disease.
 Sensitivity to UV, cerebellar ataxia, telagectasia in eyes
and lymphoreticular neoplasms are common.
 Ataxia telanectasia mutated gene is located at
chromosome 11q.
 3.FANCONI ANEMIA: Defective gene is located in
chromosome 20q and 9q.
 It is an autosomal recessive
 Defect is in repair of cross linkage damage.
 4.BLOOM SYNDROME: Mutated gene is located in
chromosome 15q.
 DNA ligase1 and Rec Q helices are defective.
 Growth retardation ,photosensitivity, lymphatic
malignancies and chromosomal breaks are seen.
 5.Hereditary Polyposis Colorectal cancer:
 The defective gene is located in chromosome 2.
 The Hmsh-1 and 2 genes involved in mismatch repair
are defective.
 This leads to hereditary predisposition COLON
CANCERS.
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