BMS 532 DNA Damage and Repair Lecture Notes PDF

DNA Damage and Repair BMS 532 BLOCK 3 LECTURE 3  Objectives 1. Explain what is meant by the term mutation and summarize how mutations can be classified (identify types of mutation and compare/contrast them) 2. Describe the consequences of mutations with particular emphasis on a...

DNA Damage and Repair BMS 532 BLOCK 3 LECTURE 3  Objectives 1. Explain what is meant by the term mutation and summarize how mutations can be classified (identify types of mutation and compare/contrast them) 2. Describe the consequences of mutations with particular emphasis on additional mutations and how they can be either more deleterious or beneficial/complementary 3. Identify the structural similarities and differences in bases with emphasis on the common and rare forms (keto vs enol & imino vs amino) and explain the role of tautomeric shift and spontaneous deamination in the generation of replication error 4. Explain the consequences for errors during replication in terms of DNA structure, future replication, DNA nucleotide sequences, and eventual protein products 5. Compare and contrast silent, missense, nonsense, and frameshift mutations in terms of changes in the DNA sequence and the corresponding protein products** 6. Compare and contrast mechanisms of endogenous DNA damage and the types of mutation induced 7. Compare and contrast mechanisms of exogenous DNA damage and the types of mutation induced by the agent including: ionizing radiation, UV damage, alkylating agents, and polycyclic aromatic hydrocarbons. 8. List the repair mechanisms and what types of damage they repair 9. Summarize the steps involved in the following repair mechanisms and explain the potential consequences of repair: Base-excision repair, nucleotide excision repair, mismatch repair, interstrand crosslink repair, homologous recombination, and nonhomologous end-joining ◦ The emphasis should NOT be on knowing each and every component of each process but rather on key molecules involved in the following key steps: recognition of damage, removal of damage, replacement of removed bases, and sealing of DNA backbone **This aim will be revisited in each  LO1 Overview of Mutation Mutation refers to a change in the base sequence of DNA ◦ Though originally believed to be entirely random (random mutation theory), it is clear there are regions more prone to mutation than others (hot spots) It is most typically applied to coding regions of genes though mutations can occur outside of coding regions and still impact function of a gene Gene mutations can be named to describe impact on genotype and phenotype ◦ Benign, pathogenic, or uncertain/unknown ◦ Additional Terms ◦ Deleterious ◦ Lethal ◦ Beneficial ◦ Conditional, etc…  LO1 Overview of Mutation (continued) Mutations can be in ◦ Germline: during embryogenesis or development/since birth or acquired and pose a risk to offspring ◦ Somatic: acquired during life with no risk to offspring Mutations can be classified as ◦ Spontaneous: occurring as a result of biological or chemical processes; most often due to replication ◦ Induced: via environmental agents or exogenous activity Additional mutations can impact the phenotypic effects of primary mutations ◦ i.e. suppressor mutations that reverse the effects of an initial mutation ◦ Intragenic suppressor = second mutation within same gene as first mutation ◦ Intergenic suppressor = second mutation is in a different gene than the first mutation LO2 ◦ These can often be thought of as changes that compensate for the original mutation The number 1 source of mutation = REPLICATION REPLICATION MACHINERY IS NOT PERFECT ORGANISMS THAT CAN WITHSTAND DRASTIC GENOMIC CHANGE HAVE LESS PRECISE MACHINERY  Base Structures Contribute to Replication Errors Keto Enol The structural similarities of bases Form Form contributes to mutation ◦ Common form of T is very similar to C ◦ Common form of G is very similar to A Keto Enol Form Form Additionally, bases can take on different structural arrangements ◦ Tautomeric Shift (movement of the proton) ◦ Further contribute to the incorporation Amino Imino errors observed following replication as Form Form they alter the hydrogen binding potential ◦ Increase probability of being missed by LO3 proofreading capabilities of the original polymerase Amino Imino LO3  Errors in Replication Overview Spontaneous mutations are rare (excluding external DNA damaging agents; 10-8 per base pair of which 99% are corrected; actual error rate = 10-10 per base pair) All polymerases exhibit the potential for errors ◦ Some contain proof-reading mechanisms that allow for nearly immediate correction of the error thus decreasing the propensity for error Replication serves as a major source of mutation predominantly through errors in base pairings ◦ Most errors in base pairings are transitions ◦ Errors in pairing can also result in transversions LO3, LO4, LO5, LO6  Errors in Replication T C TRANSITION ◦ Pyrimidine to Pyrimidine ◦ Purine to Purine ◦ Basic Change = Maintains Shape C T A G G A TRANSVERSION ◦ Pyrimidine to Purine ◦ Purine to Pyrimidine T C ◦ Basic Change = Different shape LO3, LO4, LO5, LO6 G A A few questions… How does incorporation of different base forms affect replication? (LO3) Examples: Which of the following would represent a transition mutation? The enol form of guanine would most likely pair with which of the following? A few thought questions: What if altered bases or structural changes occurred after helicase activity and immediately preceding replication? ◦ How would that impact the final products produced? ◦ Would both sets of newly formed dsDNA exhibit the same sequence after replication?  Errors in Replication Consequences Silent Mutations ◦Base substitution that still encodes for the same amino acid MISSENSE MUTATIONS ◦Gene base substitution results in one amino acid being exchanged for another LO4, LO5, LO6  Errors in Replication Consequences NONSENSE MUTATIONS ◦Create a new STOP codon resulting in a truncated protein that is almost always inactive ◦Exceptions to “functional loss” could include loss of one or more domains without loss of other domains (i.e. loss of intracellular domains of a receptor) LO4, LO5, LO6  Errors in Replication Consequences FRAMESHIFT MUTATIONS ◦Shifts the reading frame for the protein, can alter the entire amino acid sequence ◦The earlier the change in the sequence, the greater the overall change can be (from the standpoint of the number of amino acids impacted) LO4, LO5, LO6  Additional Errors in Replication Repetitive regions of DNA create unique problems during replication ◦ Strand ‘slippage’ and misalignment Deletions can occur during replication through ‘slippage’ of the template strand Duplications can occur during replication through ‘slippage’ of the newly synthesized strand Slips can be large or small LO4, LO6  LO3, LO4, Additional Types of LO6 Endogenous Damage Topoisomerase related ◦ Forms DNA lesions including suicidal complexes Spontaneous base deamination (replace amino groups with keto groups) ◦ Loss of exocyclic amine changes nucleotide ◦ Cytosine to Uracil ◦ Adenine to hypoxanthine ◦ Guanine to Xanthine ◦ 5-methyl cytosine to Thymine Abasic sites/AP sites (apurinic/apyrimidic) ◦ Loss of base with retention of backbone of DNA strand Oxidative Damage ◦ Impacts both bases and the DNA backbone LO7 Exogenous DNA Damage and Agents Ionizing Radiation (IR): ◦ Direct ◦ Impacts DNA directly generating base lesions and unique single strand breaks (3’phosphate or 3’phosphoglycolate ends) ◦ Indirect ◦ Impact on water to form hydroxyl radicals Ultraviolet Radiation (UV-A, UV-B, UV-C; direct and indirect damaging effects) ◦ Produce photoproducts ◦ Cause covalent linkages between 2 adjacent Pyrimidines ◦ Commonly cause Thymine Dimers ◦ These lesions are bulky and cause helical distortion of the DNA molecule Exogenous Chemical Agents LO7 More Common DNA Damaging Agents Alkylating Agents ◦ Affect N-glycosidic bond and generate abasic/AP sites ◦ Bifunctional agents can affect 2 different sites on DNA resulting in intra- and inter-strand crosslinks as well as DNA-protein crosslinks Aromatic Amines ◦ Form persistent lesions that eventually lead to base substitutions and frameshift mutations Polycyclic aromatic hydrocarbon (PAH) ◦ Capable of generating DNA intercalating intermediates that insert into DNA and form DNA adducts Toxins (wide range of effects) ◦ i.e. nitrous acid that causes deamination Environmental Stress (wide range of effects including mutagenesis at trinucleotide repeats) LO7 Summary Chatterjee and Walker 2017 A few more questions… How are endogenous and exogenous forms of error different? (LO6 and LO7) ◦Example: Which of the following best describes the difference between endogenous and exogenous errors in DNA? Breaks in the DNA backbone are caused by which agents? (LO7) ◦Example: Which of the following would be capable of causing a single strand break? DNA Repair FIXING ENDOGENOUS AND EXOGENOUS DAMAGE TO DNA ALL REMAINING SLIDES CORREL ATE WITH LO8 AND LO9 General Steps in Repair 1. Identify the damage 2. Remove the damage 3. Replace the removed base(s) 4. Seal the sugar backbone Mechanisms of Repair Summary Direct Repair Base Excision Repair Nucleotide Excision Repair Mismatch Repair Interstrand Crosslink Repair Homologous Recombination Nonhomologous End Joining  Summary Mechanism Types of Additional Features of Known Associated Lesions Note Disorder/Disease* BER Altered bases Only removes the damaged Linked to cognitive impairment, base neurodegenerative phenotypes, and immunodeficiency Direct Repair Altered bases Does NOT remove material but Unclear due to overlapping functions of rather chemically repairs the BER and NER base HR Double strand Requires homology and stand Cancer Predisposition and aplastic breaks invasion to generate repair anemia ICL Interstrand ICL can be repaired by more Fanconi Anemia and cancer crosslinks than one mechanism but this predisposition process is important in cancer predisposition MMR Mismatched Removes more than just one Cancer Predisposition bases base to repair the error NER Bulky, helix- Removes more than just one Xeroderma Pigmentosa and cancer distorting base to repair the error predisposition (Global) Premature aging and neurological anomalies and developmental defects (Transcription Coupled) NHEJ Double strand Can “repair” any break even*These if Cancer Predisposition, will come up again in the final block of Direct Repair Fixing an error in the DNA by direct alteration of the base Does NOT involve removal of the base or replacement but rather chemical change to fix the error Examples: Photolyase in bacteria, fungi, most plants, and some animals functions to reverse UV damage by splitting thymine dimers Alkyltransferase can reverse the effects of alkylating agents by removing methyl or ethyl groups from guanine bases Teranishi et al 2012  Base Excision Repair (BER) Primary responsibility = removal of NON-helix- distorting changes affecting individual bases Repairs small base adducts, inappropriate/oxidized bases, and single-strand breaks General Steps: ◦ DNA N-glycosylases remove base generating AP site ◦ Can be classified into 2 distinct classes based on tertiary structure ◦ Processing to cut backbone and generate free 3’ OH and 5’ Phosphate ◦ AP Endonuclease (APE) (multiple APEs exist) ◦ DNA Polymerase fills in missing base (short patch) or replaces several bases (long patch) with displacement of small fragment ◦ Reseal backbone via DNA ligase BER Details Nucleotide Excision Repair (NER) Repairs bulky helix-distorting lesions ◦ Chemically modified bases, thymine dimers, missing bases, and some crosslinks (INTRAstrand) Removes several bases and uses the remaining intact strand as a template for replacement of the missing bases Evolutionarily conserved ◦ Bacterial NER involves 4 key proteins (UvrA, UvrB, Uvr C, and Uvr D) in addition to DNA polymerase and DNA ligase ◦ Eukaryotic forms involve more proteins including XP Proteins Recognition of DNA damage in NER can be divided into 2 subpathways ◦ Global Genome (takes place at any time) ◦ Transcription Coupled (associated with increased repair of material within actively transcribed genes) Scharer Bacterial NER GGR = Global Genome Repair ◦ Not associated with transcription thus direct binding of recognition machinery without need for RNA pol association In transcription-coupled repair, the stalled transcription machinery is recognized by key proteins to facilitate the repair Transcription Coupled Repair ◦ Mfd-dependent (Mfd recognizes RNA Pol at site of stalled transcription) ◦ UvrD-dependent (UvrD binds at RNA Pol along with additional factors) After recognition, repair machinery is recruited  Eukaryotic NER General Steps: 1. Recognition 2. Opening of DNA molecule 3. Formation of Pre-incision Complex 4. Incision/Cutting of backbone upstream 5’ to the lesion 5. Initiation of repair via polymerase 6. Incision/cutting of backbone downstream 3’ of the lesion with fragment released 7. Seal backbone via DNA ligase Scharer Mismatch Repair (MMR) Highly evolutionarily conserved process for repair of mismatched bases ◦ Can be generated during replication or recombination but can also be caused exogenously Key consideration: ◦ Must determine which strand is correct and which is wrong/mismatched MutS homologs (MSH) and MutL homologs (MLH/PMS) = fundamental components for recognition General Steps ◦ Recognition of mismatched base ◦ Discernment of which strand is template strand to initiate proper repair ◦ Removal of mismatched base (and surrounding bases) via exonucleases ◦ Synthesis of replacement bases via polymerases ◦ Seal DNA backbone via DNA ligase DNA Interstrand Crosslink Repair (ICL) Specific repair for cross-linked DNA damage The damage is catastrophic as it stalls both replication and transcription Particularly important during replication at stalled replication forks Involves monoubiquitylation to change activity of key proteins and trigger repair (stabilizes FANC protein association with damage) Nucleases and polymerases are also critical to ICL repair Translesion synthesis can occur once one backbone is cleaved Can cross-over with NER and HR mechanisms for repair Deans and West 2011  Homologous Recombination (HR) Important for the repair of double strand breaks and for repair at stalled replication forks Utilizes one copy of the DNA to repair the other General Steps: ◦ Processing of break ◦ Identification of regions of homology and invasion of the strand to initiate repair (see right) ◦ Synthesis of strand using homologous template with potential recombination occurring between new DNA and template ◦ Resolution of combined units to form 2 distinct DNA double helices with the potential for a mixture of genetic material Wright et al 2018  NHEJ Important for repair of broken DNA General Steps: ◦ Recognition and processing of break ◦ Ku Heterodimer of Ku70 and Ku80 ◦ End processing and connection of the 2 broken ends via protein intermediates ◦ Role of endonucleases ◦ Sealing of the strand backbones via DNA ligase ◦ Dissociation of the repair machinery Ghosh and Raghavan 2021  Summary Mechanism Types of Additional Features of Known Associated Lesions Note Disorder/Disease* BER Altered bases Only removes the damaged Linked to cognitive impairment, base neurodegenerative phenotypes, and immunodeficiency Direct Repair Altered bases Does NOT remove material but Unclear due to overlapping functions of rather chemically repairs the BER and NER base HR Double strand Requires homology and stand Cancer Predisposition and aplastic breaks invasion to generate repair anemia ICL Interstrand ICL can be repaired by more Fanconi Anemia and cancer crosslinks than one mechanism but this predisposition process is important in cancer predisposition MMR Mismatched Removes more than just one Cancer Predisposition bases base to repair the error NER Bulky, helix- Removes more than just one Xeroderma Pigmentosa and cancer distorting base to repair the error predisposition (Global) Premature aging and neurological anomalies and developmental defects (Transcription Coupled) NHEJ Double strand Can “repair” any break even*These if Cancer Predisposition, will come up again in the final block of Even more questions… What features are similar in all forms of DNA repair? (LO8 and LO9) ◦Example: Which of the following requires strand ligation? __________ is required of all repair machinery. Additional Thought Questions: ◦What is unique about each form of repair? (LO8 and LO9) ◦Do all cell types use the same repair mechanisms at all times? (LO9) ◦Which type of repair is capable of inducing further DNA damage/mutation? (LO9)