DNA III Study guide.pdf

DNA III Mutation and repair shorthand study guide Mutations 1. Again, italics in the notes indicate supplemental or illustrative material not on the exam. 2. Study the key concepts and learning objectives carefully. 3. Mutations a. Many billions per cell per generation b. Induces spo...

DNA III Mutation and repair shorthand study guide Mutations 1. Again, italics in the notes indicate supplemental or illustrative material not on the exam. 2. Study the key concepts and learning objectives carefully. 3. Mutations a. Many billions per cell per generation b. Induces spontaneously, by DNA replication errors, by mutagens c. Must be repaired before DNA replication locks in the mutation or causes a lethal event d. Know the classes of mutagens e. Explain by uracil would not be a good alternative to thymine as the 4 th base in DNA, and why 5’-methylcytosine is mutagenic f. Understand that there are many sequence differences between individual humans, and that mutations in cancerous cells are numerous and complex. g. Know the types of mutations (silent, nonsense, missense, deletions, insertions, inversions, translocations) 4. Understand that mutations in repair pathway genes are common in cancerous cells because loss of function is the most likely form of mutation, and that loss of function of a repair gene, like loss of function of a tumor suppressor gene, is essential for progression to cancer. Also that epigenetic silencing of repair genes contributes to this progression (you don’t need to memorize specifics in the table in slide 16) 5. In slide 13, you should know names, disease, and pathways associated with mismatch repair genes, XP, BRCA1&2, Werner, Bloom, and Fanconi 6. You should understand how the Ames test works, the role of cytochrome P450 enzymes, and how to interpret a hypothetical result. Repair of mutations. 1. Know the major pathways and types of mutations they target, and associated diseases when defective. 2. Direct repair = no replacement of bases or repair polymerization…photolyase and O6- methylguanosine transferase 3. Base excision repair (BER) for repair of point mutations, base alterations. Consists of an army of DNA glycosylases to remove mismatched or damaged bases, followed by cleavage by apurinic endonuclease and repair synthesis 4. Mismatch repair (MMR) finds misincorporations not caught by DNA polymerase, recognizes the damaged daughter strand, and replaces it by repair synthesis. 1000-fold increase in replication fidelity. Defects associated with trinucleotide expansion  diseases (Lynch syndrome, Huntingtons, Fragile X. Know the diseases, not the specific repeats involved. 5. Nucleotide excision repair (NER) targets bulky damage notably thymine dimers caused by UV light. Defective components of the NER complex are associated with xeroderma pigmentosum, hence proteins a labelled XP. Some recognize the damaged site and recruit the other components, which then cleave only the damaged strand on either side, followed by repair synthesis. The complex can also be recognized by a stalled TFIIH of the transcription complex, called transcription-coupled repair. 6. Intra-strand crosslink repair (ICL) by Fanconi complex, assembles at stalled replication fork to recruit a translesion DNA polymerase. Deficient in Fanconi’s anemia. 7. ssDNA break repair by PARP (poly adenosyl ribosylase) recognizes damage, polymerizes adenosyl chain to recruit repair synthesis. Inhibitors promising in breast cancer cells if they are defective in dsDNA break repair, since ssDNA breaks that are not repaired because of the inhibitor mature into dsDNA breaks. 8. sdDNA break repair. Lethal if not repaired. a. NHEJ non homologous end joining. Ku proteins and DNA-dependent protein kinase roles. Mutagenic because of trimming to form the joint and because non- contiguous chromosomes can be joined (translocations) b. Homologous recombination uses undamaged homologous chromosome as a template to replace sequences on the damaged strand. i. Forms a Holliday junction; how this is resolved determines if a cross-over occurs of upstream and downstream sequences, or a non-cross over resolution. ii. Key players are recA (bacteria) or Rad51 (eukaryotes) which guide the invasive single stand to its complement on the homologous chromosome, and BRCA1 & 2 which facilitate this and the repair synthesis. Defects associated with breast cancer. Also Bloom’s helicase which promotes a non-crossover resolution to Holiday junctions. 9. Translesion synthesis. Not a repair mechanism in the sense that the damage is still present. But this family of alternative DNA polymerases replaces the main fork DNA polymerases when stalled by unrepaired damage, to allow the fork to bypass the mutation. Induced when DNA is heavily damaged. Mutagenic. 10. DNA damage response (DDR) is a complex pathway consisting of proteins that recognize DNA damage (ATM, ATR) a. to recruit and activate downstream proteins such ass BRCA1, p53, Cdc25, to b. block cell cycling to allow repair before replication, c. and to activate transcription of repair genes 11. Chemotherapeutic strategy exemplified by PARP inhibitors for breast cancer. Basically, inhibit whichever few repair pathways that are still active in the cancer cell to accelerate damage and death. This inhibition is less impactful on healthy cells which still have a full repertoire of repair pathways (so blocking one is less damaging).