DNA Repair

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DNA Repair

• M.Prasad Naidu
• MSc Medical Biochemistry,
• Ph.D.Research Scholar

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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 .

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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 104 or 105
  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 .

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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 .

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Single base alteration
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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 ( a d ) do not
  posses nuclease proofreading function.

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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 .

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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
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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 .

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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 .

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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 .

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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.

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

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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 .

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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.

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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 .

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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 .

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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 .

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Deamination C-U
Depurination ----gt an abasic site
Deamination of 5-mC----gtT
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DNA is damaged by Alkylation, Oxidation, and
Radiation
Often mispair with thymine GC AT
Reactive oxygen species O2-, H2O2, OH
G modification (alkylation oxidation)
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Mutations are also caused by base analogs and
intercalating agents
Base analogues
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Base excision repair pathway
(apurinic/apyrimidinic recognizes missing base)
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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 .

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

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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
  3rd to 5th phosphodiester bond 3 from the
  lesion on the 5 side the cut is some where
  between the 21st 25th 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 ( d/e in humans), ends are joined by
  DNA ligase.

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2
3
1
4
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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
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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
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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 .

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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 ) .

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contd

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

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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 .

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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 )

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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 .

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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 .

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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 .

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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.

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