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dna repair

DNA Repair

M.Prasad Naidu

MSc Medical Biochemistry,

Ph.D.Research Scholar

introduction
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 .
contd
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 .
contd1
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 .
contd2
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.
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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 .
the nature of mutations
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

abnormal regions of dna either from copying errors or dna damage are replaced by 4 mechanisms
Abnormal regions of DNA , either from copying errors or DNA damage are replaced by 4 mechanisms
  • Mismatch repair ,
  • Base excision repair ,
  • Nucleotide excision repair ,
  • Double stranded break repair .
mismatch repair
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 .
contd4
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 .
contd5
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.
slide17
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 .
clinical importance
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 .
contd7
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.
base excision repair
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 .
contd8
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 .
contd9
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 .
slide26
Deamination

C-U

Depurination

---->

an abasic site

Deamination of

5-mC---->T

dna is damaged by alkylation oxidation and radiation
DNA is damaged by Alkylation, Oxidation, and Radiation

Often mispair with thymine

G:C –A:T

Reactive oxygen species

O2-, H2O2, OH•

G modification (alkylation & oxidation)

slide29
Base excision

repair

pathway

(apurinic/apyrimidinic; recognizes missing base)

nucleotide excision repair
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 .
slide31
Incapable of base-pairing and cause the DNA

polymerse to stop during replication

Thymine dimer by ultraviolet light

contd10
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 .
contd11
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 ( δ/ε in humans), ends are joined by DNA ligase.
slide34
2

3

1

4

slide35
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

slide36
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

clinical imporatance
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 ) .
contd13
contd
  • XPA & XPC are involved in recognition & excision .XPB & XPD are helicases & interestingly are subunits of the transcription factor TFIIH .
double strand break repair
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 .
contd14
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 )
contd15
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 .
contd16
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 .
some repair enzymes are multifunctional
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 .
clinical importance1
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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