MT022 Molecular Biology and Diagnostics with Teacher Ade Start! Central Dogma of Molecular Biology Transcription DNA ➜ RNA DNA Replication DNA ➜ DNA DNA Translation RNA ➜ Protein RNA Protein Lecture 3: DNA Repair and Recombination Reference: Fix-it-Felix Objectives • To learn how the DNA is damaged and repaired. • To learn the various DNA Recombination mechanisms. • To study diseases related to dysfunctional DNA repair system. Darrion Nguyen @lab_shenanigans DNA Polymerase Proofreading DNA Damage and Repair Reference: Graham et al., 2016 https://hms.harvard.edu/news/fix Spontaneous DNA damage • Oxidative damage • Hydrolytic attack • Methylation Spontaneous DNA damage • • • Oxidative damage Hydrolytic attack Methylation Methylation Depurination • Glycosyl linkage of purine to deoxyribose hydrolyze Depurination Deamination • Conversion of nucleotide to unnatural products by hydrolysis of the amino group Deamination Deamination Deamination Pyrimidine dimers Breaks in the DNA If these DNA damages are not repaired and accumulate, then diseases will occur. DNA Double Helix is Readily Repaired Translesion polymerase • The replicative polymerase is switched out for specialized DNA polymerases and replicates past the damage. • DNA polymerase ɳ (eta) in eukaryotes Breaks in the DNA Repair of Single Strand Breaks • Mismatch Repair • Base Excision Repair • Nucleotide Excision Repair Single Strand Break DNA Repair • Mismatch Repair - genetic defects (Lynch Syndrome) • Base Excision Repair - carcinogenic exposure • Nucleotide Excision Repair - UV light (Xeroderma pigmentosum) Mismatch Repair Mismatch Repair Base Excision Repair Base Excision Repair • DNA glycosylases - “flipping out” - recognizes and removes the unnatural nucleotide • AP endonuclease - “missing tooth” - removes the sugar-phosphate backbone Nucleotide Excision Repair • “bulky lesions” - covalent reactions of DNA bases with large hydrocarbons (e.g. carcinogen benzopyrene found in tobacco smoke, coal tar, and diesel exhaust) - pyrimidine dimers caused by sunlight Nucleotide Excision Repair Nucleotide Excision Repair • • • • Excision nuclease DNA Helicase DNA Polymerase DNA Ligase Breaks in the DNA Repair of Double-strand Breaks • Nonhomologous End Joining • Homologous Recombination Non-homologous End Joining Non-homologous End Joining Reference: Graham et al., 2016 https://hms.harvard.edu/news/fix fluorescence resonance energy transfer (FRET) microscopy Homologous Recombination Homologous Recombination Homologous Recombination Homologous Recombination Homologous Recombination • Repair of broken replication fork Homologous Recombination • Repair of broken replication fork Case Study • Hereditary Non-polyposis Colon Cancer • Xeroderma Pigmentosum • BRCA1 and BRCA2 Hereditary non-polyposis colon cancer • Lynch Syndrome • Genetic disorder that causes an increased risk of developing cancer • Autosomal dominant • Mutation in the genes involved in DNA repair - Mismatch Repair Genes (MMR genes) Reference: Idos & Valle, 2004 https://www.ncbi.nlm.nih.gov/books/NBK1211/ Diagnosis Hereditary non-polyposis colon cancer • Microsatellite instability (MSI) testing Reference: Idos & Valle, 2004 https://www.ncbi.nlm.nih.gov/books/NBK1211/ Diagnosis Microsatellite instability testing by PCR Xeroderma Pigmentosum Children of the Moon Xeroderma Pigmentosum • UV radiation can cause DNA damage by triggering pyrimidine dimer formation • • Excision repair to remove dimer formation is dysfunctional Mutations in seven genes, XPA to XPG, responsible for the excision repair pathway Xeroderma Pigmentosum • Mutations in the eighth gene called XPV produce an alternate form of xeroderma pigmentosum • Affects DNA polymerase ɳ (eta) responsible for translesion synthesis Diagnosis Xeroderma Pigmentosum • Skin biopsy • Genetic testing BRCA1 and BRCA2 • Brca1 and Brca2 gene compromise the homologous recombination repair and causes hereditary breast and ovarian cancer Diagnosis Breast Cancer Summary • Discussed about the different types of DNA damage. • Learned the various ways in which DNA repair is conducted for all kinds of damage, such as dimers, single and double strand DNA breaks. • Discussed the mechanism behind DNA recombination. • Studied diseases relating to the dysfunction of DNA repair. Central Dogma of Molecular Biology Transcription DNA ➜ RNA DNA Replication DNA ➜ DNA DNA Translation RNA ➜ Protein RNA Protein Lecture 3: DNA Repair and Recombination Reference: Fix-it-Felix
