15 주차 수업자료

1. 15주차 수업자료

2. Chapter 23. Mutations and Repair

3. Types of mutation • Point mutation • Null mutation: genetic KO • Tight vs. leaky mutation • Nonsense, missense, and silent mutation • DNA sequence alteration • Base substitution • Insertion • Deletion • Inversion • Duplication • Translocation • Base substitution • Transition: Py  Py, Pu  Pu • Transversion: Py  Pu, Pu  Py • Segregation of base alteration

4. Missense mutation • Conservative substitution • Radical replacement • Temperature sensitive (ts) mutation • Correct folding at normal low (permissive) temperature • Misfolding or unfolding at high (restrictive) temperature • Conditional mutation

5. Nonsense and deletion mutation • Nonsense mutation = chain termination mutation • Deletion mutation can be lethal

6. Insertion mutation • Insertion mutation • Due to mobile genetic element • Transposon/retrotransposon • Polarity effect • Gene activation? • By mutagenic chemicals or mistakes of DNA polymerase

7. Frameshift mutation • Frameshift mutation destroys the function of protein

8. Inversion, translocation, and duplication • Intragenic: severe effect • Intergenic: mild effect

9. Chemical mutagens • Induced mutation (by chemicals, radiation, heat) vs. spontaneous mutation • Base structure alteration • Base analog • Intercalation by base pair analog

10. Mutation by radiation • Ionizing radiation (high energy; far UV, X-ray, gamma-ray) vs. non-ionizing radiation (low energy; near UV, visible, IR, microwave, radio wave) • Thymine dimer formation by UV • Repair by photoreactivation • Transcription-coupled excision repair • Recombination repair by RecA • SOS error-prone repair by DNA polymerase V (no proofreading subunit)

11. Spontaneous mutation • DNA replication error rate in bacteria: 1/10,000,000 (20 times more in lagging strand than in leading strand… why?) • Insertion or omission of bases due to the slippage of DNA polymerase • Occurs where there are multiple repeats of a short sequence • Example: human trinucleotide repeat expansion disease (Huntington’s disease)

12. Mutation due to mispairing Inversion

13. Spontaneous mutation due to tautomerization and deamination • Other spontaneous mutation • Oxidative damage: 8-hydroxy-guanine • Non-enzymatic methylation (S-adenosyl-methionine): 3-methyl-adenine

14. DNA repair: mismatch repair

15. DNA repair: mismatch repair

16. DNA repair: nucleotide excision repair • “cut and patch” repair • Not as sensitive as mismatch repair system • Repair damage due to UV radiation (thymine dimer) • UvrAB binding  UvrC replaces UvrA  nick by UvrC and UvrB  UvrD (helicase) unwinds damaged DNA  degradation of damaged DNA and gap filling by pol I (5’-exo and pol activity)  nick sealing by DNA ligase

17. DNA repair: base excision repair • Removal of altered base by DNA glycosylase  AP site  AP endonuclease  free 3’-OH group  pol I (5’-exo/pol)  ligase • In case of oxidized G: removal of oxoG by MutM glycosylase, removal of A (only when found opposite 8-oxoG) by MutY glycosylase  AP site  same as above

18. DNA repair: alkylated base repair • Suicide demethylase • Ada (adaptation to alkylation) protein: dual role demethylase • methyl group removal from altered base (Ada C-term)  degradation of Ada • methyl group removal from phosphate backbone (Ada N-term)  Ada becomes transcriptional activator

19. DNA repair: photoreactivation Photolyase

20. DNA repair: recombination by RecA

21. DNA repair: SOS error-prone repair by DNA polymerase V Photolyase

22. DNA repair: transcription-coupled excision repair

23. DNA repair: double-strand break repair in eukaryotes • Non-homologous end joining • Double-strand break by ionizing radiation  Ku protein  DNA-dependent protein kinase (DNA-PK)  phosphorylation and activation of XRCC4  DNA ligase IV

24. Reversion Mutation in codon 50: GAA (Glu, -)  AAA (Lys, +) True revertant: AAA  GAA in codon 50 Second-site revertant: mutation in codon 25 (+  -) • True revertant: restoration of original base sequence • Second-site revertant: second base change (suppressor mutation) cancels out the effect of first one • Intragenic suppression vs. extragenic suppression

25. Nonsense suppression • Suppression of nonsense mutation by tRNA alteration (suppressor tRNA) • Stop codon • Amber (UAG) • Ochre (UAA) • Opal (UGA) • Amber suppressor tRNA • Ochre suppressor tRNA

26. Chapter 24. Recombination

27. Homologous vs. non-homologous recombination • Breakage  crossover  rejoining

28. Homologous recombination: molecular basis • Holliday junction: 2 heteroduplex region • Formation of patch recombinants or hybrid DNA by resolvase (RuvC) – isomerization possible through chi intermediate • Holiday junction migration by RuvA (bind junction) and RuvB (drives migration)

29. Homologous recombination: how? • Crossover at chi site • RecBCD • RecA: single strand invasion

30. Non-homologous recombination: site-specific recombination • Lambda attachment site: attBOB’, att POP’ • After integration, hybrid lambda attachment site: attBOP’, att POB’ • Int protein: integrase • Xis protein: excisionase

31. Recombination in eukaryotes

32. Gene conversion • Conversion of one allele to the other during meiosis by mismatch repair  non-Mendelian ratio among progeny • Ascospore in Ascus: Mendelian ratio from a single zygote