DNA Repair

M.Prasad Naidu

MSc Medical Biochemistry,

Ph.D.Research Scholar

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• 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 .

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 .

Single base alteration

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.

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 mutationsSimple 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

1) Mismatch repair ,

2) Base excision repair ,

3) Nucleotide excision repair ,

4) Double stranded break 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 .

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 .

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.

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 .

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

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 .

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 .

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

• 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 .

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 .

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 .

Deamination

C-U Depurination ---->an abasic site

Deamination of 5-mC---->T

DNA is damaged by Alkylation, Oxidation, and Radiation

Often mispair with thymine G:C –A:T

Reactive oxygen speciesO2-, H2O2, OH•

G modification (alkylation & oxidation)

Mutations are also caused by base analogs and intercalating agents

Base analogues

Base excision repairpathway

(apurinic/apyrimidinic; recognizes missing

base)

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

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 .

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 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.

1

3

4

2

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

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 • 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 .

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 ) .

contd

• 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 generation .

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

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 )

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 .

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 • 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 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.