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MOLECULAR BIOLOGY I
Dr. Sana Khurshid
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DNA Repair
• A cell generally has only one or two sets of genomic DNA.
Damaged proteins and RNA molecules can be quickly
replaced by using information encoded in the DNA, but DNA
molecules themselves are irreplaceable.
• Maintaining the integrity of the information in DNA is a
cellular imperative, supported by an elaborate set of DNA
repair systems.
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DNA Damage
• Changes in the DNA sequence result from
1. DNA synthesis, errors
• including incorrect base-pairing or insertion of one to a
few extra nucleotides
2. environmental insults
• chemicals, for example, nitrous acid,
• radiation, for example, ultraviolet light , and high energy
ionizing radiation,
3. Spontaneously altered or lost
• mammalian DNA altered or lost spontaneously at a rate of
many thousands per cell per day
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• Most such spontaneous changes in DNA are temporary

because they are immediately corrected by processes
collectively called DNA repair.
• Only rarely do the cell's DNA maintenance processes fail and
allow a permanent change in the DNA.
• The most serious outcome is a change in the base sequence
of the DNA, which, if replicated and transmitted to future
cell generations, becomes permanent.
• A permanent change in the nucleotide sequence
• of DNA is called a mutation.
• and it can destroy an organism if the change occurs in a
vital position in the DNA sequence.
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DNA repair Systems

1. Direct repair

2. Excision repair
A. Methyl-directed mismatch repair
B. nucleotide excision repair
C. base excision repair

3. Repair of double-strand breaks

 Damaged Bases Can Be
Directly Repaired
Called DIRECT REPAIR
In a few cases, the covalent modifications of
nucleotides can be reversed by specific enzymes
– Photolyase can repair thymine dimers induced by UV
light
It splits the dimers restoring the DNA to its original
condition

– O6-alkylguanine alkyltransferase repairs alkylated

bases
It transfers the methyl or ethyl group from the
6
base to a cysteine side chain within the
alkyltransferase protein
Direct repair of damaged bases in DNA

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• The original pyrimidine bases remain in DNA, now restored

to their normal state. As might be expected from the fact
that solar UV irradiation is a major source of DNA damage
for diverse cell types, the repair of pyrimidine dimers by
photoreactivation is common to a variety of prokaryotic and
eukaryotic cells, including E. coli, yeasts, and some species
of plants and animals. Curiously, however, photoreactivation
is not universal; many species (including humans) lack this
mechanism of DNA repair.

• O6-methylguanine methyltransferase Enzymes are

widespread in both prokaryotes and eukaryotes, including
humans.
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Excision Repair
• Although direct repair is an efficient way of dealing with
particular types of DNA damage, excision repair is a more
general means of repairing a wide variety of chemical
alterations to DNA. Consequently, the various types
of excision repair are the most
important DNA repair mechanisms in both prokaryotic
and eukaryotic cells. In excision repair, the
damaged DNA is recognized and removed, either as free
bases or as nucleotides. The resulting gap is then filled in by
synthesis of a new DNA strand, using the undamaged
complementary strand as a template. Three types
of excision repair—base-excision repair, nucleotide-
excision repair, and mismatch repair—enable cells to cope
with a variety of different kinds of DNA damage.
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A. Methyl-directed mismatch repair

• Sometimes replication errors escape the proofreading
function during DNA synthesis, causing a mismatch of one
to several bases.
• 1. Identification of the mismatched strand:
• In E. coli , the Mut proteins identify the mispaired
nucleotide(s). MutHLS system
• 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 exact mechanism by which the daughter strand is
identified in eukaryotes is not yet known.
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• 2. Repair of damaged DNA:

• When the strand containing the
mismatch is identified, an
endonuclease nicks the strand
and exonuclease removes
nucleotides at the 5'- and 3'-ends
of the mismatch
• The gap left by removal of the
nucleotides is filled, using the
sister strand as a template, by a
DNA polymerase.
• the newly synthesized DNA is
joined to the remaining stretch of
the original DNA strand by DNA
Ligase
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B. Repair of damage caused by ultraviolet

(UV) light
• Exposure of a cell to UV light can result in the covalent joining of
two adjacent pyrimidines (usually thymines), producing a dimer.
• These thymine dimers prevent DNA polymerase from replicating
the DNA strand beyond the site of dimer formation.
• Thymine dimers are excised in bacteria by UvrABC proteins in a
process known as nucleotide excision repair.
• A related pathway involving XP proteins is present in humans.
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• Recognition and excision of

dimers by UV-specific
endonuclease:
• First, a UV-specific endonuclease
(called uvrABC excinuclease)
recognizes the dimer, and cleaves
the damaged strand on both the
5'-side and 3'-side of the dimer.
• A short oligonucleotide
containing the dimer is released,
leaving a gap in the DNA strand
that formerly contained the
dimer. This gap is filled by DNA
polymerase and ligase.
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UV radiation and cancer

• Pyrimidine dimers can be formed in the skin cells of humans
exposed to unfiltered sunlight. In the rare genetic disease
xeroderma pigmentosum (XP), the cells cannot repair the
damaged DNA, resulting in extensive accumulation of
mutations and, consequently, early and numerous skin
cancers. XP can be caused by defects in any of the several
genes that code for the XP proteins required for nucleotide
excision repair of UV damage in humans.
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C. Correction of base alterations (base

excision repair)
1. The bases of DNA can be
altered, either
• spontaneously, as is the case with
cytosine, which slowly undergoes
deamination (the loss of its amino
group) to form uracil,
Or
• by the action of deaminating or
alkylating compounds. For example,
nitrous acid, is a potent compound that
deaminates cytosine.
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2. 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
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• 1. Removal of abnormal bases:

• Abnormal bases, such as uracil are
recognized by specific
glycosylases that hydrolytically
cleave them from the
deoxyribose–phosphate backbone
of the strand.
• This leaves an apyrimidinic site
(or apurinic, if a purine was
removed), both referred to as AP
sites.
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• 2. Recognition and repair of an AP

site:
• Specific AP-endonucleases
recognize 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, base-free, sugar
phosphate residue.
• A DNA polymerase and DNA ligase
complete the repair process.
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D. Repair of double-strand breaks

• The repair pathways considered to this point generally work
only for lesions in double-stranded DNA, the undamaged
strand providing the correct genetic information to restore
the damaged strand to its original state.

• However, in certain types of lesions, such as double strand

breaks, double-strand cross-links, or lesions in a single-
stranded DNA, the complementary strand is itself damaged
or is absent.
• High-energy radiation or oxidative free radicals can cause
double- strand breaks in DNA, which are potentially lethal to
the cell.
• Such breaks also occur naturally during gene
rearrangements.
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• There are two possible avenues for repair:

1. recombinational DNA repair /homologous recombination
repair
or,
1. when lesions are unusually numerous, error-prone
repair/ nonhomologous end-joining repair
• The first is nonhomologous end-
joining repair, in which the ends
of two DNA fragments are brought
together by a group of proteins
that effect their re-ligation.
However, some DNA is lost in the
process. Consequently, this
mechanism of repair is error prone
and mutagenic.
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• The second repair system,

homologous recombination
repair, uses the enzymes that
normally perform genetic
recombination between
homologous chromosomes
during meiosis. This system is
much less error prone than
nonhomologous end-joining
joining because any DNA that
was lost is replaced using
homologous DNA as a
template.
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Figure description
• A schematic for DSB repair by HR and NHEJ pathways. DSBs
can be repaired by either HR or NHEJ. For initiation of HR,
DSB ends must be resected to expose 3′ overhangs of ssDNA
by the exonuclease activity. The resulting ssDNA–RAD51
presynaptic filaments are capable of invading the
homologous region in the nearby duplex DNA, forming a
triplex DNA called a D-loop. DNA polymerases further
extend DNA synthesis.
• NHEJ directly seals two DSB ends and does not generally
require DSB end resection. Binding of Ku at DSB ends
recruits other proteins, which subsequently joins two
broken DNA ends.