DNA Repair

• Mutation


  Change in the normal base pair sequence
• Small scale mutations


  • Base substitutions / modifications
  • Deletions
  • Insertions
• Deletions


  One or more nucleotides are eliminated from DNA sequence (frameshift)
• Insertions


  Addition of one or more base pairs into the DNA sequence (frameshift)
• Types of DNA lesions

  • Abasic (without a base) – an excision of a base
  • Apurinic/apyrimidinic sites or AP sites
  • AP sites can block transcription and replication
• Purine bases


  • G and A
• Pirimidine bases


  • C, T and U
• Spontaneous DNA lesions


  • Oxidation
  • Hydrolysis
  • Methylation
• Nucleoside alterations


  • Alkylation (gain of an alkyl group), oxidation (gain of an oxygen atom), deamination (loss of an amine group), methylation (methyl group is added), hydrolysis (a molecule of water is added)
• Human cells lose about 18000 purine bases (adenine and guanine) every day
• Depurination can release guanine (G) as well as adenine (A) from DNA
• The major type of deamination reaction converts cytosine to uracil. Uracil is a demethylated form of thymine
• How chemical modifications of nucleotides produce mutations
• Types of DNA lesions

  • Single base alterations
  • Bulky lesions (abnormal covalent bonds between nearby nucleotides, chemical compounds bind covalently to the DNA)
• Thymine dimers


  • Covalent link between the two adjacent T bases to form a dimer
• Replication errors


  • Mismatches-nucleotide is miss-incorporated
• Interstrand crosslinks


  • Abnormal covalent bonds may be formed between complementary strands of the DNA
• Three general classes of the DNA repair mechanisms

  • Direct repair
  • DNA excision repair: base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR)
  • Double-strand break repair (DSB): homologo
• DNA repair


  1. Genetic information can be stored stably in DNA sequences only because of the large set of DNA repair enzymes continuously scan the DNA and replace any damaged nucleotides

  2. BER: the altered base is removed by DNA glycosylase enzyme followed by excision of the sugar-phosphate backbone

  3. NER: a small section of the DNA strand surrounding the damage is removed from DNA

  4. In NER and BER, the gap left at the DNA is filled by DNA polymerase and DNA ligase

  5. MMR is used to repair the mistakes created during the replication, and it detects the mismatch between non-complementary base pairs. MutS proteins are bound to the mismatch base pair while MutL is scanning the nearby DNA for a cut

  6. NHEJ resells the accidental double strand breaks

  7. Elevated level of DNA damage can cause the delay in the cell cycle, which ensures that DNA damage is repaired before a cell divides
• Double strand breaks (DSB) repair


  1. Non-homologous DNA end joining (NHEJ): DS ends are degraded and not replaced, found in non-dividing cells, preferred mechanisms during G0-G1, high potential for loss of genetic information (deletions, insertions, and translocations)

  2. Homologous recombination: exchange of DNA strands between the pair of homologous duplex DNA, repairing DNA breaks produced by UV irradiation or reactive chemicals, one of the most efficient DNA repair mechanisms in cells, highly conserved in all cells
• Homologous recombination


  Takes place only between the homolog DNA duplexes, accurate repair of DNA double strand breaks, creates new combination of the genetic material, assures accurate chromosome segregation, occurs just after replication
• Each chromosome contains a single strand of DNA. A dyad is a pair of chromosomes. The dyad is held together by the centromere, which has about 1-10 million bases of DNA, mostly repetitive DNA, compacted and with no transcription. Kinetochore contains about 80 proteins and must be broken to separate chromatids
• Homologous recombination steps


  1. DSB occurs

  2. Sister chromatids pair

  3. Degradation of the 5’ ends

  4. Strand exchange

  5. Branch point migration and DNA synthesis: DNA helix is reformed

  6. DNA ligation
• Strand invasion


  Catalysed by RecA (in bacteria) / RAD51 (in eukaryotes). Protein-DNA filament, Invading strand, Match
• Wilson and Elledge: 'Science 2002'
• BRCA2 in HR


  Double-strand breaks (DSBs), The binding of replication protein A (RPA) to the damaged end. RPA recruits BRCA2 to the sites of DNA damage. BRCA2 binds to Rad51 and helps to bring it to the sites of damage
• Inherited mutations in BRCA2 can increase the breast cancer risk by 90% and ovarian cancer risk by 15-20%. Increased chance of developing these cancers at a young age. Increased risk of melanoma, lymphoma, colon cancer...
• BRCA2 is located at Ch13, a tumour suppressor gene
• Abnormal covalent bonds may be formed between complementary strands of the DNA

  Links
• Replication and environment factors can result in DNA mutations
• Three general classes of the DNA repair mechanisms

  • Direct repair
  • DNA excision repair: base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR)
  • Double-strand break repair (DSB): homologous end joining (HEJ) and non-homologous end joining (NHEJ)
• Most of DNA lesions could be repaired by different mechanisms
• Direct repair


  • Rapid process
  • Do not need template
  • Do not involve breakage of the phosphodiester backbone
  • Energetically is very costly to the cells and it is used to quickly repair DNA lesions
  • MGMT (O-6-methylguanine-DNA methyltransferase) acts by reversing the damaged base
• Base excision repair (BER)

  Uses specific glycosylase enzymes to remove abnormal bases - endonuclease and phosphodiesterase to cut sugar backbone, then gap is filled and sealed
• Nucleotide excision repair (NER)


  Repairs bulky lesions that distort the helix. Removal of a lesion leaves a gap which is repaired
• Mismatch repair (MMR)


  Repair mistakes during the DNA replication
• Mismatch repair (MMR) have four steps

  1. Recognition of the damage

  2. Cleavage by endonuclease

  3. Removal by nuclease or helicase activity

  4. Replacement by polymerase and ligase activity
• Non-homologous DNA end joining (NHEJ)

  1. DS ends are degraded and not replaced

  2. It is found in non-dividing cells

  3. Preferred mechanisms during G0-G1

  4. High potential for loss of genetic information (deletions, insertions, and translocations)
• The molecular basis and disease relevance of non-homologous DNA end joining


  Zhao et al., Nat Rev Mol Cell Bio, 2020
• DNA damage delays progression of the cell cycle


  • DNA repair enzymes delay the progression of the cell cycle until DNA repair is complete
  • Progression of the cell cycle is stopped if DNA damage has been detected
  • Mammalian cells can have G1 cell cycle arrest (transition from G1 to S), slow down S phase, and G2 cell cycle arrest (transition from G2 to M)