BB1725 Lecture 5 DNA Damage and Repair PDF

BB1725 Biology of the Cell Lecture 5: DNA Damage and Repair Dr Joseph Hetmanski [email protected] Today’s Lecture The lecture today will: look at DNA damage and repair By the end of the lecture, you should be able to explain: How DNA can be damaged How DNA can be repa...

BB1725 Biology of the Cell Lecture 5: DNA Damage and Repair Dr Joseph Hetmanski [email protected] Today’s Lecture The lecture today will: look at DNA damage and repair By the end of the lecture, you should be able to explain: How DNA can be damaged How DNA can be repaired Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 2 Mutations A mutation is any change from the normal DNA sequence: Deletions (loss of nucleotide(s)) Insertions (gain of nucleotide(s)) Substitutions (change of one base for another) Transitions Purine to purine - e.g. A to G Pyrimidine to pyrimidine - e.g. C to T Transversions Purine to pyrimidine (or vice versa) Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 3 Mutations A mutation is any change from the normal DNA sequence High numbers of mutations in the germ line would destroy the species High numbers of mutations in somatic cells would destroy the individual organism (e.g. human) DNA is a macromolecule subjected to damage from many sources Therefore, methods of DNA repair are needed Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 4 Exogenous (External) Damage Ionising radiation (e.g. X rays) Single or double-strand breaks in DNA UV (sunlight) Thymine cross-linking Chemicals Hydrocarbons in cigarette smoke Aflatoxins in mouldy peanuts Base modification and cross-linking between bases Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 5 Endogenous (Internal) Damage 1. DNA replication errors 2. Hydrolysis - water drives the reaction Spontaneous depurination Spontaneous deamination 3. Oxidation Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 6 Thymine cross-linking Two base alteration UV light induced thymine-thymine dimers (strong covalent bonds) If not repaired, one Figure 5–39 The ultraviolet radiation in sunlight can cause the formation of thymine dimers thymine may be deleted Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. on replication Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 7 Hydrolysis: Deamination and depurination Depurination: The removal of guanine or adenine Deamination: Usually, the conversion of cytosine to uracil Other types can occur No break to the DNA Figure 5–38 Depurination and deamination are the most frequent backbone spontaneous chemical reactions known to create serious DNA damage in cells Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 8 Hydrolysis: Deamination Loss of amino group Cytosine to uracil Adenine to hypoxanthine 5 methylcytosine to thymine Figure 5–40A Chemical modifications of nucleotides, if left unrepaired, produce mutations Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 9 Hydrolysis: Depurination Loss of adenine or guanine bases Each nucleated human cell loses ~5,000 DNA purines per day Loss of pyrimidines is only 5% loss of purines Figure 5–40B Chemical modifications of nucleotides, if left unrepaired, produce mutations Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 10 Oxidation Normal metabolic by- products of respiration Reactive oxygen species (ROS) - “Free Radicals” Superoxide - O2 - Hydroxyl - OH Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 11 Oxidative Damage ROS attack purine and pyrimidine rings More than 100 oxidative modifications of DNA: Guanine to 8-hydroxyguanine Pairs with A instead of C Helical distortion Additional covalent bonding between base and sugar- phosphate backbone Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 12 What are the consequences of DNA damage? Cell death Functional decline of tissues Organ issues/failure (Neurodegeneration, kidney injury, cardiovascular disease) Cancer Developmental deficiencies Embryonic lethality Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 How often does DNA damage occur? DNA: Damage and Repair Mechanisms in Humans, Ambekar et al., Glob J Pharmaceu Sc, 2017 How are we even still here? … DNA repair mechanisms Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 DNA Repair Mechanisms Proofreading activity of DNA polymerase Direct repair Reverses the DNA damage Base Excision Repair (BER) Removes single damaged bases Single-strand break repair Main protector against metabolism damage Nucleotide Excision Repair (NER) Removes Thymine dimers and large chemical adduct Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 15 DNA Repair Mechanisms (cont.) Mismatch Repair (MMR) Removes mismatched base pairs and insertion/deletion loops Non-homologous end joining (NHEJ) DS break repair Homologous recombination repair (HR) DS break repair Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 16 Direct Repair Infrequently used O6-methylguanine-DNA methyltransferase 3 genes implicated 6 O -methylguanine-DNA me-O6 | methyltransferase 5’ G 3’ 3’ 5’ Removes methyl groups C O6-methylguanine-DNA methyltransferase G 5’ 3’ 3’ 5’ C Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 17 Base Excision Repair (BER) DNA glycosylases identify and remove the damaged base AP endonuclease and phosphodiesterase cut the sugar- phosphate backbone Gap is filled by DNA polymerase b Nick is sealed by DNA ligase Figure 5–41A A comparison of two major DNA repair pathways. Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 18 Nucleotide Excision Repair (NER) Damaged bases are detected Helicases and nucleases act to open up and cut either side of the mutated bases 24-32 bp oligonucleotide is removed Gap is repaired by DNA polymerase e or d Figure 5–41B A comparison of two major DNA repair pathways Nick is sealed by DNA ligase Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 19 Compare and contrast… Figure 5–41 A comparison of two major DNA repair pathways Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 20 Mismatch Repair (MMR) MMR proteins correct mismatched base pairs or small insertion or deletion loops caused by errors in DNA replication MSH proteins form ‘sliding clamps’ that move along the DNA to identify damage MLH proteins are targeted by MSH and come in to recognise mismatched DNA and initiate repair Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 21 Mismatch Repair (MMR) Four major steps: 1. Mismatch recognition 2. Recruitment of additional MMR factors 3. Search for signal identifying the incorrect (newly synthesised) strand and degradation of this before the mismatch 4. Resynthesis of excised DNA https://youtu.be/HYS6EKnQcv0 Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 22 Non-homologous end joining (NHEJ) Corrects double strand breaks (DSB) Two broken ends are joined regardless of sequence More error prone (some loss or gain of DNA) Bit of a ‘sledgehammer’ approach Figure 5–45A Cells can repair double-strand breaks in one of two ways Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 23 Homologous recombination repair (HR) Corrects double strand breaks (DSB) Similar to homologous recombination during meiosis Needs the presence of sister chromatids (replicated DNA) Single strand of DNA from donor sister chromatid invades the damaged sister chromatid Figure 5–45B Cells can repair double-strand breaks in one DNA repair is very accurate of two ways Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 24 Compare and contrast Figure 5–45 Cells can repair double-strand breaks in one of two ways Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 25 DNA Damage Response 1. DNA damage detected 2. Arrest of cell cycle Damage tolerance Or Damage repair Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 26 Common Themes of DNA Repair Detect the damage - proteins detect and bind Remove the damage - nucleases etc. Resynthesis/Repair - DNA polymerases and ligases Regulation - protein kinases etc. Use correct reference if possible Effects/Outcomes: Accurate repair = survival Failure to repair = cell death Misrepair = mutation, genomic instability Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 27 Defective Repair Mechanisms Base excision repair Homologous Recombination repair colon cancer Bloom syndrome (premature Nucleotide excision repair ageing) UV sensitivity NHEJ repair Xeroderma pigmentosa - Leukaemia skin disorder DNA damage response and repair Mismatch repair ATM - Ataxia telangiectasia Hereditary nonpolyposis BRCA1 BRCA2 - breast cancer colon cancer - HNPCC P53 – many types of cancer Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 28 BRCA1 case study Breast cancer type 1 susceptibility protein Involved in both checkpoint arrest (green arrows) and double stranded DNA repair (red arrows) Homologous end joining Mismatch repair Non-homologous end joining Females with an abnormal BRCA1 or BRCA2 gene have up to an 80% risk of developing breast cancer by age 90 Such a high risk factor that women are screened for BRCA mutations Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 29 BRCA1 case study single-nucleotide polymorphism Deletion/insertion which causes frameshift mutations 17q21 Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 Summary DNA Damage – exogenous/endogenous DNA Repair: Direct repair Base Excision Repair (BER) Nucleotide Excision Repair (NER) Mismatch Repair (MMR) Non-homologous end joining (NHEJ) https://www.nature.com/news/dna-repair-sleuths-win-chemistry-nobel-1.18515 Homologous recombination repair (HR) Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 31 References Explore the material for this week on Brightspace Molecular Biology of the Cell by Alberts et al. 7th Ed. - Chapter 5 Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 32 Additional links of interest DNA Damage & Repair: Mechanisms for Maintaining DNA Integrity: https://www.nature.com/scitable/topicpage/dna- damage-repair-mechanisms-for-maintaining-dna- 344/ DNA: Damage and Repair Mechanisms in Humans: https://juniperpublishers.com/gjpps/pdf/GJPPS.MS.I D.555613.pdf Brunel University London – BB1725 Biology of the Cell – Lecture 5 – 2024/25 33 Questions?