DNA Damage and it's Repair

Introduction :

• DNA damage refers to any alteration or breaks in DNA that may be cause by various factors including radiations, mutagenic chemicals, or error during replications and finally results in mutations. 
• DNA Damage Repair refers to any cellular process of fixing those alterations or breaks in DNA.

DNA DAMAGE

• DNA damages may be caused by Hydrolytic damages or, Radiations or, Mutagens.

A. By Hydrolytic Damages

1. Deamination

• Deamination of Cytosine to form unnatural Uracil

• Deamination of Adenine to form Hypoxanthine that base pairs with Cytosine.

• Deamination of Guanine to form Xanthine that base pairs with Cytosine.

• Deamination of 5-methyle-cytosine to form Thymine.

2. Depurination

• It is caused by spontaneous hydrolysis of N-Glycosyl Linkage.
• It produces an abasic site (Deoxyribose, lacking a base) that causes Transcriptional silencing.

B. By Alkylation

• Alkylating agents(methyl or ethyl groups) like N-methyl-N¹-nitro-N-nitrosoguanidine are transferred to reactive sites on bases and phosphates.

• In case of Guanine, C-6 of Guanine is most vulnerable to alkylation, which results in forming O⁶ methyl guanine, that base pairs with Thymine.

C. By Oxidation

• Reactive Oxygen-species like O₂- , H₂O₂-, OH- cause oxidation of Guanine to form 7,8-dihydro-8-oxoguanine (oxoG).
• The oxoG base pairs with Adenine (which is most common in human cancer)
• It can base pair with Cytosine as well.

D. By Radiations

1.By UV Rays

• Photochemical fusion of two Pyrimidines that occupy adjacent to each other on the same polynucleotide chain to form a dimer. E.g. T-T Dimer.

2.By Gamma-rays and X-rays

• The Gamma or X-rays break the dsDNA either by directly attacking or by generating reactive oxygen species.

E. By Base analogs

• 5-Bromo uracil is an analog of Thymine that mispairs with Guanine.

F. By intercalating agents

• Intercalating agents are polycyclic flat rings that bind to DNA bases and cause the Deletion or Insertion of base pairs.
• Examples of Intercalating agents include Proflavin, Acridine, and Ethidium.

DNA DAMAGE REPAIR

A. Direct reversal

• Here the repair enzyme simply reverses the damage.

E.g. 1 Photoreactivation

• It involves the direct reversal of pyrimidine dimers.
• The Photolyase enzyme receives the energy from UV rays and break the covalent bonds of adjascent pyrimidines.

E.g. 2 Removal of methyl groupp.

• Removal of methyl group from O⁶ methyl guanine by Methyl transferase.

B. Excision Repair

i.Base Excision Repair

• It is the most common way of repairing the damaged DNA.
• This method involves the hydrolysis of glycosidic bond by Glycosylase enzyme, followed by removal of abasic sugar from the DNA backbone by endonucleolytic cleavage.
• After successful removal of damaged nucleotide, a repair DNA Polymerase & DNA Ligase restore intact DNA strands using the undamaged strand as a template.

Glycosylase Enzymes are specific

• One recognizes Uracil (the result of Cytosine deamination).
• Another removes oxoG (the result of Guanine oxidation).
• A total of 8 different DNA Glycosylases have been identified in the nuclei of human cells.
• These enzymes diffuse laterally along the minor grooves of the DNA until a specific lesion is detected and remove oxoG.
• The damaged base of major groove is flipped out of the double helix and enters into the specific pockets of the Glycosylase enzyme.
• Removes T in T:G Mismtch that arises from the spontaneous deamination of 5 Methyl-Cytosine to establish normal C-G pairing.
• If the damaged base is not removed by excision before replication, e.g. in case of oxoG, the Glycosylase enzyme recognizes oxoG:A base pair and removes Aii.Nucleotide Excision Repair
• Nucleotide excision repair enzymes do not recognize any particular lesion, they only recognize distortions to the DNA double helix.
• The removal of a nucleotide stretch creates a single strand gap in DNA filled by polymerase using undamaged strand as template.
• a. In E. coli.
  • It involves 4 Proteins- UvrA, UvrB, UvrC & UvrD.
  • UvrA- UvrB Complex Scans the DNA.
  • UvrA detects distortion to the helix.
  • UvrB melts the DNA to create a single strand, attaches to it  and recruits UvrC.
  • UvrC creates 2 incisions- one located 8-Nucleotides away on 5' side and the second, located 4 or 5- nucleotides away on 3' side to create a 12-13 base pairs long single stranded DNA segment, i.e. accessible to helicase, UvrD.
  • Finally, DNA Polymerse I and Ligase fill in resulting gap to form an intact DNA.

  

  

  

• b. In Higher Cells

  • XPC Protein detects distortion to the helix.
  • XPA & XPD Protins melt the DNA to create a single strand.
  • RPA Proteins bind to unwound single strands.
  • ERCC1-XPF creates incision on 5' side.
  • XPG creates incision on 3' side.
  • The resulting DNA is 24-32 nucleotide long.
  • Finally, DNA Polymerse β and Ligase fill in resulting gap to form an intact DNA.

  

Mutations in the human Uvr genes, the XP Genes result into a disease called Xeroderma pigmentosum, characterized by Extreme sun sensitivity, leading to a very high risk of skin cancer.

C. Recombination Repair

• It occurs when 2 strands of the DNA are broken at the same time.
• It is accomplished by double strand break repair pathway by retrieving informations from homologous chromosome.

• Non Homologous End Joining (NHEJ) Pathway 
  • In case if the sister chromosome has not ben generated, the NHEJ Pathway occurs.
  • Here single strand tails are removed by nucleases and gap filled by polymerases.
  • This pathway is mediated by KU  Proteins, that align the ends of broken chromosome & recruit other repair proteins called KU Mediated NEHJ Proteins.
  • It is an inefficient process allowing survival of only one in a thousand yeasts.

  

  
  

  
  

D. Translesion DNA Synthesis

• Sometimes, DNA Polymerase encounters lesions like Pyrimidine dimer or an apurinic site, that has not been repaired, due to which the polymerse ceases the replication.
• In E.coli,  the Translesion DNA Synthesis is carried out by certain proteins called UmuC and UmuD.
• UmuC is a member of Y-family of DNA polymerase.
• The UmuC incorporates nucleotides independently of base pairing. 
• Due to this independent base pairing, this mechanism is highly error prone.

E. Transcription coupled Repair

• Here, the RNA Polymerase serves as damage sensing protein.
• In Eukaryotes, the transcription factor, TFIIH unwinds the DNA, that has two subunits- XPA & XPD.

Reference:

Watson, J.D., Baker, T.A., Bell, S.P., Gann, A., Levine, M. & Losick, R. (2014) Molecular biology of the gene. 7th ed. Pearson Education.