DNA Damage, Repair and Clinical Significance: Biochemistry for Medics Lecture Notes, Study notes of Genetics

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DNA Damage, RepairDNA Damage, Repair and Clinical and Clinical significance significance By- Professor (Dr.) Namrata Chh abra Biochemistry for Medics- Le cture Notes

IntroductionIntroduction

• (^) DNA in the living cell is subjected to many chemical alterations.
• (^) The genetic information encoded in the DNA has to remain uncorrupted
• (^) Any chemical changes must be corrected.
• (^) A failure to repair DNA produces a mutation. 2 Biochemistry for Medics-Lecture

Agents that DamageAgents that Damage DNA DNA

• (^) Chemicals in the environment o (^) Aromatic hydrocarbons, including some found in cigarette smoke o (^) Plant and microbial products, e.g. the Aflatoxin produced in moldy peanuts o (^) Chemicals used in chemotherapy, especially chemotherapy of cancers. 4 Biochemistry for Medics-Lecture

Types of DNA damageTypes of DNA damage S.N o. Type of Damage Examples

1. Single-base alteration A.Depurination B.Deamination of cytosine to uracil C.Deamination of adenine to hypoxanthine D.Alkylation of base E.Insertion or deletion of nucleotide F.Base-analog incorporation
2. Two-base alterations A. UV light–induced thymine-thymine (pyrimidine) dimer B. Bifunctional Alkylating agent cross-linkage
3. Chain breaks A. Ionizing radiation B. Radioactive disintegration of backbone element C. Oxidative free radical formation
4. Cross-linkage (^) A. Between bases in same or opposite strands B. Between DNA and protein molecules (eg,^5 Biochemistry for Medics-Lecture

A) Direct DamageA) Direct Damage Reversal Reversal The direct reversal of DNA damage is by far the simplest repair mechanism that involves a single polypeptide chain, with enzymatic properties which binds to the damage and restores the DNA genome to its normal state in a single-reaction step. The major polypeptides involved in this pathway are: i) DNA photolyases, the enzymes responsible for removing cyclobutane pyrimidine dimers from DNA in a light-dependent process called as photo reactivation 7 Biochemistry for Medics-Lecture

A) Direct DamageA) Direct Damage Reversal Reversal ii) O6-methylguanine- DNA methyltransferase I and II (MGMT), also called DNA- alkyltransferases , remove the modified bases like O6-alkylguanine and O4-alkylthymine.

• (^) The photolyase protein is not found in all living cells. However, the DNA- alkyltransferases are widespread in nature. 8 Biochemistry for Medics-Lecture

i) Base Excision Repairi) Base Excision Repair (BER) (BER)

• (^) BER is initiated by DNA glycosylases, which catalyze the hydrolysis of the N- glycosidic bonds, linking particular types of chemically altered bases to the deoxyribose-phosphate backbone.
• (^) DNA damage is excised as free bases, generating sites of base loss called apurinic or apyrimidinic (AP) sites. 10 Biochemistry for Medics-Lecture

i) Base Excision Repairi) Base Excision Repair (BER) (BER)

• (^) The AP sites are substrates for AP endonucleases.
• (^) These enzymes produce incisions in duplex DNA as a result of the hydrolysis of a phosphodiester bond immediately 5' or 3' to each AP site.
• (^) The ribose-phosphate backbone is then removed from the DNA through the action of a specific exonuclease called deoxy ribophosphodiesterase or dRpase.
• (^) Finally, the DNA polymerase and a ligase catalyze the incorporation of a specific deoxyribonucleotide into the repaired site, enabling correct base pairing 11 Biochemistry for Medics-Lecture

ii) Nucleotide excisionii) Nucleotide excision repair (NER) repair (NER)

• (^) This mechanism is used to replace regions of damaged DNA up to 30 bases in length.
• (^) Common causes of such DNA damage include ultraviolet (UV) light, which induces the formation of cyclobutane pyrimidine-pyrimidine dimers, and smoking, which causes formation of benzo[ a ]pyrene-guanine adducts.
• (^) Ionizing radiation, cancer chemotherapeutic agents, and a variety of chemicals found in the environment cause base modification, strand breaks, cross- linkage between bases on opposite strands or between DNA and protein, and numerous other defects.
• (^) These are repaired by a process called nucleotide excision-repair 13 Biochemistry for Medics-Lecture

ii) Nucleotide excisionii) Nucleotide excision repair (NER) repair (NER)

• (^) NER is a much more complex biochemical process than BER, especially in eukaryotic cells.
• (^) Several gene products are required in a multiple step process, during which the ordered assembly of DNA proteins provides an enzymatic complex that discriminates damaged from undamaged DNA. 14 Biochemistry for Medics-Lecture

ii) Nucleotide excisionii) Nucleotide excision repair (NER) repair (NER)

• (^) In Escherichia coli there are three specific proteins, called UvrA, B and C, involved in lesion recognition and endonuclease incision.
• (^) This fragment is released by UvrD helicase action, generating a gap that is finally submitted to repair synthesis 16 Biochemistry for Medics-Lecture

Transcription-CoupledTranscription-Coupled NER NER

• (^) Nucleotide-excision repair proceeds most rapidly in cells whose genes are being actively transcribed on the DNA strand that is serving as the template for transcription.
• (^) If RNA polymerase II, tracking along the template (antisense) strand), encounters a damaged base, it can recruit other proteins, to make a quick fix before it moves on to complete transcription of the gene. 17 Biochemistry for Medics-Lecture

iii) Mismatch repairiii) Mismatch repair (MMR) (MMR)

• (^) 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.
• (^) An exonuclease then digests this strand from the GATC through the mutation, thus removing the faulty DNA. This can occur from either end if the defect is bracketed by two GATC sites.
• (^) This defect is then filled in by normal cellular enzymes according to base pairing rules 19 Biochemistry for Medics-Lecture

iii) Mismatch repairiii) Mismatch repair (MMR) (MMR)

• (^) This mechanism corrects a single mismatch base pair (eg, C to A rather than T to A) or a short region of unpaired DNA.
• (^) The defective region is recognized by an endonuclease that makes a single-strand cut at an adjacent methylated GATC sequence.
• (^) The DNA strand is removed through the mutation, replaced, and religated. 20 Biochemistry for Medics-Lecture