DNA repair: DR, MMR and BER

direct repair

• simple - energetic advantage

• safe - minimise chance of mistakes introduced by repair process

• 3 examples known:

1. repair of single-strand breaks

2. repair of pyrimidine dimers

3. repair of methyl groups

single-stranded DNA breaks

• can arise from DNA replication, recombination and repair, exposure to x rays or gamma radiation

• arise from removal of bases during base excision repair

• usually not a problem for non-dividing cells but if cell carries out replication 1 arm of homologous chromosome is lost

• breaks that occur close to eachother in opposing strands or in replication of a single strand can generate double-stranded break - more dangerous

single-stranded break repair

• DNA ligase joins 3’-OH and 5’ phosphate group of adjacent nucleotides by phosphodiester bond

• larger gaps cannot be repaired, cannot join free nucleotides

• bacterial DNA ligases use NAD as energy source, eukaryotic DNA ligases use ATP as energy source

repair of pyrimidine dimers by DNA photolyase

• pyrimidine dimer in DNA induced by UV light - induces covalent bond formation between adjacent pyrimidine residues

• DNA photolyase enzyme forms complex with DNA dimer

• light harvesting molecules folate and flavin subunits of DNA photolyase

• folate absorbs blue light and transfers it to flavin - flavin excited to reduced state (FADH^. - neutral radical) from original state (FADH-)

• flavin (FADH^.) breaks apart dimer by donating an electron - forms unstable pyrimidine dimer radical anion which spontaneously breaks apart

• once dimer broken apart, electron is transferred back to flavin from other parts of the enzyme or surrounding protein residues so that it is returned to its reduced state and regains its catalytic activity

repair of O⁶-methylguanine by direct repair

• O⁶-methylguanine mispairs with thymine

• methyltransferase (Ada) detects distortion in DNA backbone due to mispairing 

• methyltransferase has cysteine residue - cysteine residue accepts methyl group so that it is removed from O⁶-methylguanine

• works best on double stranded DNA

Ada protein structure and activity

• regulates set of genes involved in repairing alkylating damage - N terminal of Ada switches on adaptive response once it has been methylated

• N terminus accepts methyl groups from backbone, C terminus accepts methyl groups from O⁶ methylguanine

• once modified, inactivated protein targeted for degradation - cannot be reactivated

mismatch repair: proof-reading

• bases look normal so not detected by other repair mechanisms

• bacterial DNA polymerase II backtracks (3’ to 5’ direction) and excises incorrectly inserted nucleotide through activity of dnaQ

mismatch repair in E coli

• Dam methylase adds methyl group to adenine in the GATC sequence - allows enzymes to identify strand which needs correcting (newly synthesized strand)

• MutS recognises mismatch by slight distortion in base pairing and MutL assembles to form a complex. MutH binds to the GATC site.

• DNA is threaded through MutHLS complex in a loop until MutH encounters hemimethylated DNA

• MutH cleaves unmethylated strand at the hemimethylated GATC site. The nicked strand is unwound by MutU and an exonuclease degrades the daughter strand beyond the position of the mismatch

• repair synthesis takes place using DNA polymerase (I, II or III) to replace nucleotides by DNA synthesis and the nick is sealed by DNA ligase

base excision repair

• modified base excised and entire nucleotide replaced

• specialized in fixing single-base lesions containing small chemical modifications

• DNA glycosylases recognize the type of damaged base and cleave the bond between the base and sugar backbone

• AP endonucleases recognize abasic site and cleave phosphodiester backbone on either 3’ or 5’ side

• second enzyme removes deoxyribose sugar from backbone so there is a gap

• single nucleotide gap filled by DNA polymerase and nick sealed by DNA ligase

double strand break repair

• DNA double helix broken by double strand break

• double strand breaks undergo nuclease degradation - gives duplex with 3’ ended single stranded tails

• one single strand invades a second duplex at region of homology - forms a displacement loop as strand from duplex displaced

• 3’ end of invading strand acts as primer for DNA synthesis

• displacement loop translocates across new strand until single stranded section complementary to other section is synthesised

• newly synthesised strand joins