DNA Repair

DNA repair mechanism

1. DNA Repair
M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2. Introduction
•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 .

5. contd
•Physiological conditions strongly favors the amino
& lactam forms , the unfavored tautomers may
participate in mutagenic events if they were
unrepaired .
•The equilibrium where by one tautomer is more
stable than another is only about 10
4
or 10
5
in
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 .

6. contd
•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 .

7. Single base alteration

8. contd
•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
molecule .
•The analogous mammalian enzymes ( α &
δ ) do not posses nuclease proofreading
function.

9. contd
•Replication errors occurs even with efficient
repair system lead to the accumulation of
mutations.
•Damage to DNA occurs by environmental ,
physical & chemical agents classified to 4
types .

11. The nature of mutations
Simple mutations:
Transitions(pyrimidine-to-pyrimidine and
purine-to-purine)
Transversions(pyrimidine-purine and
purine-to-pyrimidine)
Insertions and deletions (a nucleotide or a
small number of nucleotides)

★
point mutations: mutations that alter a single
nucleotide

12. 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 .

13. Mismatch 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 .

14. contd
•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
GATC .

15. contd
•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
sites .
•The defect is filled by normal cellular
enzymes according to the base pairing rules.

17. 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
strand .

18. MutS scans the DNA, & recognize the mismatch or
the distortion in the DNA backbone .

20. Clinical importance
•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
HNPCC .

21. contd
•Another gene hMLH1 is associated with most other
cases .
•hMLH1 gene is human analogue of bacterial
mismatch repair gene Mut L .
•Microsatellites are repeated sequences of DNA.

•These repeated sequences are common, and
normal.
•The most common microsatellite in the humans is a
dinucleotide repeat of CA, which occurs tens of
thousands of times across the genome .

22. contd
•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.

23. 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 .

24. contd
•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 .

25. contd
•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 .

26. Deamination
C-U
Depurination
---->
an abasic site
Deamination of
5-mC---->T

27. DNA is damaged by Alkylation,
Oxidation, and Radiation
Often mispair with thymine
G:C –A:T
Reactive oxygen species
O
2-
, H
2
O
2
, OH•
G modification (alkylation & oxidation)

28. Mutations are also caused by base
analogs and intercalating agents
Base
analogues

29. Base excision
repair
pathway
(apurinic/apyrimidinic; recognizes missing
base)

30. 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 .

31. Thymine dimer by ultraviolet light
Incapable of base-pairing and cause the DNA
polymerse to stop during replication

32. contd
•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 .

33. contd
•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 3
rd

to 5th phosphodiester bond 3 ‘ from the lesion & on
the 5’ side the cut is some where between the 21
st
&
25
th
bond .
•Thus a fragment of 27 – 29 nucleotides long is
exicised .
•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.

34. 1
3
4
2

35. 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

36. Transcription coupled
DNA repair:
nucleotide excision
repair system is
capable of rescuing
RNA polymerase that
has been arrested by
the presence of lesions
in the DNA template

38. Clinical Imporatance
•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 .

39. contd
•The inherent defect seems to involve the
repair of damaged DNA , particularly thymine
dimers .
•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 ) .

40. contd
•XPA & XPC are involved in recognition &
excision .XPB & XPD are helicases &
interestingly are subunits of the transcription
factor TFIIH .

41. 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
generation .
•Some chemotherapeutic agents destroy cells
by causing double stranded breaks or
preventing their repair .

42. contd
•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 )

43. contd
•DNA – PK has a binding site for DNA free
ends & another for ds DNA just inside these
ends .
•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 .

44. contd
•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
pairs .
•The extra nucleotide tails are removed by an
exonuclease & the gaps are filled and closed
by DNA ligase .

46. 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
transcription .
•Another component of TFIIH is involved in
cell cycle regulation .
•Thus three critical cellular processes may be
linked through use of common proteins .

47. Clinical importance
•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.