BBMB 405 1nd Edition Lecture 26Outline of Last Lecture XIV. Chapter 28: DNA Replication, Repair and RecombinationC. DNA replication is highly coordinatedD. Many types of DNA damage can be repairedOutline of Current Lecture XIV. Chapter 28: DNA Replication, Repair, and RecombinationD. Many types of DNA damage can be repairedCurrent LectureXIV. Chapter 28: DNA Replication, Repair, and RecombinationD. Many types of DNA damage can be repaired1. Proofreading: Pol III contains exonuclease site to chew back mutated DNA strand, the mutation creates change in interactions and structure so genome is unstable, detects mismatches because of distortions in double helix, 2. Mismatch repair (MMR): a. MutS finds lesions (damage) by finding distortions in helixb. MutL assists MutS and recruites endonuclease MutHc. MutH recognizes newly synthesized strandd. Cleave the DNA then resynthesizee. How correct strand identified (bacteria)? Parental strand is methylated so it identifies the unmethylated strand, when one strand is methylated and one is not it is called hemimethylation, in humans this is not fully understoodf. Why called mut genes? Because if they are not present mutations are far more likely to occur3. Oxidation: Guanine to 8-oxoguaninea. Oxidation by reactive oxygen speciesb. Mismatches with adenine through Hoogsteen base pairingThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.c. If 8-oxoguanine binds in same conformation as guanine then there is steric clash, but if inverts then will only be able to create two hydrogen bonds so bonds with Adenine4. Deamination: Adenine to hypoxanthinea. Hypoxanthine is more likely to pair with cystosineb. Switch base pair from AT to GC5. Alkylation: Aflatoxin B1a. Aflatoxin is fungal metabolite and a health concern if it is in food sourcesb. Upon digestion cytochrome P450 converts aflatoxin into active DNA modifying agentc. Active DNA-modifying agent adds something to the base which opens up helix so helix has 12 to 13 base pairs per turn6. Hydrolysis: Remove glycosidic bond to form apurinic or apyrimidinic (AP) sitesa. Remove base from sugarb. Depurination can occur spontaneouslyc. Both depurination and depyrimidination occur as intermediates during base excision repair7. Base-excision Repair (BER)a. Recognition: DNA glycosylases recognize specific lesionsb. Extrudes base: cleaves glycosidic bondc. DNA nicking by AP endonucleased. Removal of dRP by deoxyribose lyasee. Fill gap by DNA pol and DNA ligase8. Deamination of Cytosine: converts cytosine to uracil which can also lead to damage, leads to GC to AT mutations9. UV light DNA damage: a. Create thymine dimer activates double bonds and links bonds to create cyclobutaneb. DNA photolyase: not in humans, convert energy from light to reverse damage, cofactors MTHF10. Nucleotide excision repair (NER)a. Excision of incorrect nucleotide fragment by UvrABC excinuclease:- UvrA: DNA lesion recognition- UvrB: DNA binding and UvrC recruitment- UvrC: cleaves DNA 4 nt downstream and 8 nt upstream of DNA damageb. UvrD: DNA helicase II removes excised segmentc. DNA polymerase I resynthesizes DNA and displaces remaining components of UvrABCcomplexd. DNA ligase seals final nick11. Interstrand crosslinks: a. Psoralen: stack within base pairs then by UV light forms bonds with bases of both strandsb. Very hard to removec. Generally block replication and lead to arrest of cell division: could use to treat cancer12. DNA damage can be used therapeutically: nitrogen mustard connects two adjacent basesin interstrand linkages, chemotherapeutics13. Ames Test:a. Mutation in the histadine producing genes so histadine must be added.b. Some can reverse mutation and make histadinec. Add liver to culture so the compound would be metabolized like it would in the body; they did this to better be able to see how the mutation would effect the cell in real life14. Huntington’s disease: Slippage event occurs where polymerase pauses causing DNA to melt and reanneal incorrectly and DNA to be repaired incorrectly15. Xeroderma pigmentosum: Mutation in NER enzyme, can’t repair UV damaged
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