MSc Medical Biochemistry,
• The maintenance of the integrity of the
information in DNA molecules is of utmost
importance to the survival of the species .
• The major responsibility for the fidelity of
replication resides in specific pairing of
nucleotide bases .
• Proper pairing is dependent upon the
presence of favoured tautomers of the purine
& pyrimidine nucleotides .
• Physiological conditions strongly favors the amino
& lactam forms , the unfavored tautomers may
participate in mutagenic events if they were
• The equilibrium where by one tautomer is more
stable than another is only about 104
favor of that with great stability.
• The favoring of preferred tautomers & the proper
base pairing could be ensured by monitoring the
base pairing for 2 times .
• Double monitoring appear in both mammalian &
bacterial systems .
• First monitoring occurs at the time of insertion of
the deoxyribonucleoside triphosphates , & later by
a follow up ,energy requiring mechanism which
removes all improper bases that may occur in the
newly formed strand .
• Unfavored tautomers occur more frequently than
once in every 10 8
– 10 10
base pairs .
• The mechanisms responsible for DNA repair
in E .coli include the 3’ to 5’ exonuclease
activities of one of the subunits of
polymerase III complex & of the polymerase I
• The analogous mammalian enzymes ( α &
δ ) do not posses nuclease proofreading
• Replication errors occurs even with efficient
repair system lead to the accumulation of
• Damage to DNA occurs by environmental ,
physical & chemical agents classified to 4
The nature of mutations
Insertions and deletions (a nucleotide or a
small number of nucleotides)
★point mutations: mutations that alter a single
Abnormal regions of DNA , either from
copying errors or DNA damage are replaced
by 4 mechanisms
1) Mismatch repair ,
2) Base excision repair ,
3) Nucleotide excision repair ,
4) Double stranded break repair .
• Mismatch repair corrects errors made when
DNA is copied , for example a Cytosine could
be inserted opposite an A , or the polymerase
could slip or stutter & insert 2 – 5 extra
unpaired bases .
• Specific proteins scan the newly synthesized
DNA , using adenine methylation within
GATC sequence as the point of reference .
• The template strand is methylated & newly
synthesized strand is not methylated .
• This difference allows the repair enzymes to
identify the strand that contains the errant
nucleotide which requires replacement .
• If a mismatch or small loop is found , a
GATC endonuclease cuts the strand bearing
the mutation at a site corresponding to the
• An exonuclease digests this strand from
GATC through the mutation thus removing
the faulty DNA .
• The above digestion can occur from either
side if the defect is bracketed by 2 GATC
• The defect is filled by normal cellular
enzymes according to the base pairing rules.
In E .coli three proteins ( Mut S , Mut L & Mut H )
are rrequired for recognition of the mutation &
nicking of the strand . Other cellular enzymes
ligase , polymerase & SSBs remove & replace the
MutS scans the DNA, & recognize the mismatch or
the distortion in the DNA backbone .
• Faulty mismatch repair is linked to hereditary
nonpolyposis colon cancer ( HNPCC ) .
• Genetic studies linked HNPCC in some families to
a region of chromosome 2 .
• The gene on chromosome 2 is hMSH2 is human
analogue of Mut S protein that is involved in
mismatch repair .
• Mutations of hMSH2 account for 50 - 60 % of
• Another gene hMLH1 is associated with most other
• hMLH1 gene is human analogue of bacterial
mismatch repair gene Mut L .
• Microsatellites are repeated sequences of DNA.
• These repeated sequences are common, and
• The most common microsatellite in the humans is a
dinucleotide repeat of CA, which occurs tens of
thousands of times across the genome .
• Muted hMSH2 & hMLH1 mismatch repair
enzymes results in increased size of
microsatellites , this must affect the function
of a protein critical in surveillance of the cell
cycle in these colon cells .
• The appearance of abnormally long or short
microsatellites in an individual's DNA is
referred to as microsatellite instability.
• Microsatellite instability (MSI) is a
condition manifested by damaged DNA due
to defects in the normal DNA repair process.
Base Excision Repair
• This mechanism is suitable for replacement
of a single base but is not effective at
replacing regions of damaged DNA .
• The depurination of DNA which happens
spontaneously due to the thermal lability of
the purine N – glycosidic bond , occurs at a
rate of 5000 – 10,000 /cell / day at 37 ° C .
• Cytosine , adenine & Guanine bases in DNA
spontaneously form uracil , hypoxanthine or
xanthine respectively .
• None of the above are normal bases .
• N – glycosylases can recognize these
abnormal bases & remove the base itself
from the DNA .
• This removal marks the site of the defect &
allows an apurinic or apyimidinic
endonuclease to excise the abasic sugar .
• The proper base is replaced by repair , DNA
polymerase & the ligase returns the DNA to
its original state , this series of events is
called base excision repair .
• By similar series of steps involving initially
the recognition of the defect , alkylated
bases & base analogues can be removed
from DNA .
an abasic site
DNA is damaged by Alkylation,
Oxidation, and Radiation
Often mispair with thymine
Reactive oxygen species
, H2O2, OH•
G modification (alkylation & oxidation)
Mutations are also caused by base
analogs and intercalating agents
(apurinic/apyrimidinic; recognizes missing
Nucleotide Excision Repair
• This mechanism is used to replace regions
of damaged DNA up to 30 bases in length .
• UV light induces the formation of
cyclobutane pyrimidine – pyrimidine dimers .
• Smoking causes formation of benzopyrene –
guainine adducts .
Thymine dimer by ultraviolet light
Incapable of base-pairing and cause the DNA
polymerse to stop during replication
• Ionizing radiation , cancer chemotherapy &
chemicals found in environment cause base
modification , strand breaks , cross – linkage
between bases on opposite strand or
between DNA protein & numerous other
defects are repaired by this mechanism .
• Nucleotide excision repair is complex
process involves more gene products than 2
other types of repair , essentially involves
hydrolysis of 2 phosphodiester bonds on the
strand containing the defect .
• A special excision nuclease ( exinuclease )
consisting of at least 3 sub units in E .coli & 16
polypeptides in humans .
• In eukaryotic cells the enzymes cut between the 3rd
to 5th phosphodiester bond 3 ‘ from the lesion & on
the 5’ side the cut is some where between the 21st
• Thus a fragment of 27 – 29 nucleotides long is
• After the strand is removed it is replaced by exact
base pairing through the action of polymerase ( δ/ε
in humans), ends are joined by DNA ligase.
1.UvrA and UvrB scan DNA to identify a distortion
2. UvrA leaves the complex,and UvrB melts DNA
locally round the distortion
3. UvrC forms a complex with UvrB and creates
nicks to the 5’ side of the lesion
4. DNA helicase UvrD releases the single stranded
fragment from the duplex, and DNA Pol I and ligase
repair and seal the gap
repair system is
capable of rescuing
RNA polymerase that
has been arrested by
the presence of lesions
in the DNA template
• Xeroderma pigmentosum is an autosomal
recessive genetic disease .
• The clinical syndrome include marked
sensitivity to sunlight ( UV rays ) with
subsequent formation of multiple skin
cancers & premature death .
• The risk of developing skin cancer is
increased 1000 to 2000 fold .
• The inherent defect seems to involve the
repair of damaged DNA , particularly thymine
• Cells cultured from patients with xeroderma
pigmentosum exhibit low activity for the
nucleotide excision repair process .
• Seven complementation groups have been
identified using hybrid cell analysis so at
least 7 gene products ( XPA – XPAG ) .
• XPA & XPC are involved in recognition &
excision .XPB & XPD are helicases &
interestingly are subunits of the transcription
factor TFIIH .
Double Strand Break Repair
• The repair of double strand breaks is part of
the physiological process of immunoglobulin
gene rearrangement .
• It is also important mechanism for repairing
damaged DNA such as occurs as result of
ionizing radiation or oxidative free radical
• Some chemotherapeutic agents destroy cells
by causing double stranded breaks or
preventing their repair .
• Two proteins are involved in the
nonhomologous rejoining of a ds break .
• Ku , a hetero dimer of 70 & 86 kDa subunits ,
bind to free DNA ends & has latent ATP
dependent helicase activity .
• The DNA bound Ku hetero dimer recruits an
unusual DNA dependent Protein kinase
( DNA – PK )
• DNA – PK has a binding site for DNA free
ends & another for ds DNA just inside these
• It allows the approximation of the 2
separated ends .
• The free end DNA/Ku/DNA – PK complex
activates the kinase activity in the later .
• DNA – PK reciprocally phosphorylates Ku &
the other DNA – PK molecule on the
opposing strand , in trans .
• DNA – PK then dissociates from the DNA &
Ku, resulting in activation of the Ku helicase.
• This results in unwinding of the 2 ends .
• The unwound approximated DNA forms base
• The extra nucleotide tails are removed by an
exonuclease & the gaps are filled and closed
by DNA ligase .
Some repair enzymes are multifunctional
• DNA repair proteins can serve other
purposes example some repair enzymes
found as components of the large TFIIH
complex that play a central role in gene
• Another component of TFIIH is involved in
cell cycle regulation .
• Thus three critical cellular processes may be
linked through use of common proteins .
• In patients with ataxia telangiectasia ,an
autosomal recessive disease characterized
by cerebellar ataxia & lymphoreticular
neoplasms , in these patients there appears
to exist an increased sensitivity to damage by
X rays .
• Fanconis anemia an autosomal recessive
anemia characterized by an increased
frequency of cancer & by chromosomal
instability , probably have defective repair of
cross linking damage.