Molecular biologyMechanism of repair

B. C. MuthubharathiI M.Sc., Biotechnology

DNA Damage – A short note

Both physical and chemical agents have the ability to alter the primary structure of DNA DNA susceptible to environmental damages – Exogenous, Endogenous

Ionizing radiation Exposure to chemicals UV rays Cell’s metabolic products Absorption of thermal energy during metabolism – Tautomeric shift (rearrangement of bases) Leads to alteration, Mutation etc.,

Tautomeric shift

Rearrangement of basepairs occur during normal metabolism Results in the production of structural isomers of a base Enhance the mispairing

But the system is lucky

Repair mechanism1. Automatic 2. Proteins and enzymes are available ProcessI. Excision / removal of damaged sectionII. Reconstruction of complementary removed section

Types of repair mechanism Excision repair mechanisma. Nucleotide excision repairb. Base excision repairc. Mismatch excision repair Photo reactivation Recombinational repair SOS response Adaptive response Heat shock repair

Nucleotide excision repair Cut – patch mechanism Bulky lesions like thymine dimers can be removed and rearranged Steps1. Recognition of bulky lesion in DNA2. Hydrolyzing the phosphodiester bond in deoxyribose backbone in 5’ side of lesion3. Excising the lesion ( along with a limited number of nucleotide on its 3’ end)4. Filling the resultant gap with information given by complementary strand5. Closing the nicked DNA to generate intact strand

Pathways

Transcription coupled pathwaytemplate strand is repairedoccur during transcriptionlesion is signaled by RNA polymerase

Global genomic pathwayless efficientremaining of DNA is repairedmuch slow

(1) damage recognition in the global pathway is mediated by an XPC-containing protein complex, whereas damage

recognition in the transcription-coupled pathway is thought to be

mediated by a stalled RNA polymerase in conjunction with a CSB protein

(2) DNA strand separation (by XPB and XPD proteins, two helicase subunits of TFIIH

(3) incision (by XPG on the 3’ side and the XPF–ERCC1 complex on the

5’ side) (4) excision, (5) DNA repair synthesis (by DNA polymerase ) (6) ligation (by DNA ligase I).

NER in E. Coli ABC excinuclease ( Uvr A, Uvr B, Uvr C) A complex A2B ( Uvr A, Uvr B) which scans the

DNA for any damage and binds to the lesion Uvr A is dissociated and leaving Uvr B – DNA

complex Uvr C binds to Uvr B 12-13 nucleotides are removed by Uvr D

helicase The gap is filled by DNA polymerase I and DNA

ligase

Base Excision Repair DNA glycosylase recognize DNA lesion and remove the N-glycosyl

bond This cleavage creates apurine or apyrimidine site (AP site or abasic

site) Several DNA glycosylase are found and each specific for a particular

type of DNA lesions DNA lesions may because of

uracil formation( deamination of cytosine)8-oxoguanine3-methyladenine (transfer of methyl group)

Example for BER REPAIR OF 8-OXOGUANINE BY DNA GLYCOSYLASE• Inspection of G-C pair on the damaged DNA by DNA glycosylase• When the enzyme come across the oxoGC, it inserts a specific aminoacid

chain to the DNA which creates a 180 of rotation. • Then the base fits to the active site of enzyme and cleaved form

associated sugar• Once the altered purine or pyrimidine is removed the deoxyribose

phosphate is remaining in the site and it is excised by AP endnuclease• Polymerization by DNA polymerase • Final sealing is done by DNA Ligase III

Mismatch repair Mismatched basepair causes a distortion in the geometry double

helix Also called Methyl directed post replication repair system The mismatches are nearly always corrected to reflect the

information in the old (template) strand, so the repair system must somehow discriminate between the template and the newly syrrthesized strand

Steps • Tagging the template DNA with methyl group to differentiate from

newly synthesized DNA• Methylation is done in N6- position of adenine in 5’ GATC sequence

• The newly synthesized strand is repaired according to the information stored in the methylated parent strand

• Mismatch repair system efficiently repair nearly 1000bp in hemi methylated strand

• MutL and MutS proteins form a complex and binds to the mismatched basepair • MutH protein binds to the MutL. MutH has a site specific endonuclease activity

Proteins involvedMutL, MutS, MutHDNA helicase IIexonuclease VII/ Rec J nucleaseexonuclease I / exonuclease X

Photoreactivation DNA damages like pyrimidine dimer formation due to UV radiation DNA photolyase enzymes are involved The enzyme use energy utilized from absorbed light to reverse the damage Photolyase has 2 cofactors – MTHF PolyGlu (N5,N10 –

methenyltetrahryodfolylpolygultamate FADH