1. F mechanism of DNA replication F practice questions LECTURE 13: DNA: STRUCTURE & REPLICATION II

2. MECHANISM OF DNA REPLICATION F DNA replication requirements... 1. H bonds between bases must be broken 2. chain separation/unwinding 3. available pools of 4 dNTPs: A = T, C  G 4. enzymes F E. coli... $ ~  4.7 × 10 7 bp (47,000,000) $ molecule has ~ 4.7 × 10 6 turns (10 bp/turn) $ 1500 bp/s, ~ 20 min to divide with 1 origin

3. MECHANISM OF DNA REPLICATION FDNA polymerase I (pol I)... several types of activity 1. polymerase catalyzes chain growth 5'  3' 2. exonuclease removes mismatches 3'  5' 3. exonuclease degrades double stranded DNA 3'  5'

4. MECHANISM OF DNA REPLICATION FDNA polymerase III (pol III) F... holoenzyme of 20+ subunits 1. polymerase catalyzes chain growth 5'  3'

5. MECHANISM OF DNA REPLICATION Fprokaryotes have a single origin of replication Fbidirectional replication with 2 forks  single terminus F~ 20 minutes (*... book says 40)

6. MECHANISM OF DNA REPLICATION F E. coli origin of replication Ftandem repeat consensus sequence Fprotein binding sites

7. MECHANISM OF DNA REPLICATION Freplication to completion...

8. MECHANISM OF DNA REPLICATION Feukaryotes have multiple origins of replication Fbidirectional replication with 2 forks F1.4 h (yeast) to 24 h (cultured animal cells)... or more Fissues... $linear vs circular $coordination of multiple chromosomes Fpulse/chase and autoradiography...

9. MECHANISM OF DNA REPLICATION Feukaryotes have multiple origins of replication Fbidirectional replication with 2 forks F1.4 h (yeast) to 24 h (cultured animal cells)... or more Fissues... $linear vs circular $coordination of multiple chromosomes Fpulse/chase and autoradiography... Drosophila

10. MECHANISM OF DNA REPLICATION FDNA structure Freplication Fenzymes Ffunctions

11. MECHANISM OF DNA REPLICATION FDNA structure Freplication $5'  3' Fenzymes Ffunctions 3'3' 5'5' 3'3' 5'5'

12. MECHANISM OF DNA REPLICATION FDNA structure Freplication $5'  3' $phosphodiest er bond Fenzymes Ffunctions

13. MECHANISM OF DNA REPLICATION Fhelicases disrupt H-bonds binding strands of DNA double helix

14. MECHANISM OF DNA REPLICATION Fsingle-stranded binding proteins (SSBs) stabilize DNA and prevent formation of duplex

15. MECHANISM OF DNA REPLICATION Ftopoisomerases relax supercoiling of DNA as it unwinds... e.g., DNA gyrase

16. MECHANISM OF DNA REPLICATION Ftopoisomerases relax supercoiling of DNA as it unwinds... e.g., DNA gyrase

17. MECHANISM OF DNA REPLICATION FDNA gyrase - catalyzed supercoiling

18. MECHANISM OF DNA REPLICATION FDNA polymerase catalyzes replication 5'  3'

19. MECHANISM OF DNA REPLICATION FDNA polymerase catalyzes replication 5'  3' Fextends but cannot initiate replication...

20. MECHANISM OF DNA REPLICATION FRNA primase enzyme

21. MECHANISM OF DNA REPLICATION Fpriming synthesis Fprimase enzyme Fsynth. short (~30 bp) RNA primer Fprimase + DNA + proteins = primosome Fenzymes Ffunctions

22. MECHANISM OF DNA REPLICATION Fprimase + DNA + proteins = primosome

23. MECHANISM OF DNA REPLICATION Fleading & lagging strand Freplication is 5'  3' Freplication moves along template 3'  5' FDNA pol III most synthesis FDNA pol I fills gaps on lagging strand FDNA ligase seals gaps

24. MECHANISM OF DNA REPLICATION Freplication fork Flagging strand a) RNA primer growth b) DNA elongation as Okazaki fragments c) RNA primer removed by DNA pol I d) DNA ligation

25. MECHANISM OF DNA REPLICATION Fdimeric DNA pol III holoenzyme model

26. MECHANISM OF DNA REPLICATION Fchromosome ends Fleading strand has no problem... Flagging strand cannot add RNA primer at end... leaving gaps Ftelomeres = DNA repeats made by... Ftelomerase

27. MECHANISM OF DNA REPLICATION Ftelomerase has RNA template for making telomeres Ftelomere-specific DNA probe (yellow)

28. MECHANISM OF DNA REPLICATION Fenzyme summary...

29. MECHANISM OF DNA REPLICATION Fyou should know names and functions of: $topoisomerase ( e.g., DNA gyrase) $single-stranded binding proteins (SSBs) $DNA helicase $DNA pol I $DNA pol III $DNA pol III holoenzyme $RNA primer $RNA primase $ primosome $ Okazaki fragment $ DNA ligase $ telomerase

30. PRACTICE QUESTIONS you should be able to do CH7, p. 250 # 1 - 25