1. Figure 8.2 Objectives: Identify the key molecular players involved in DNA replication Construct a sequence of events that summarizes the process of DNA replication Compare and contrast leading strand versus lagging strand synthesis DNA Replication

2. Polymer of nucleotides: adenine, thymine, cytosine, guanine Double-stranded/Double helix "Backbone" of each strand is deoxyribose-phosphate Strands held together by hydrogen bonds between AT and CG Strands are antiparallel DNA: A Review Figure 8.4 What do we already know about DNA?

3. LE 16-9_4 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. Each “ daughter ” DNA molecule consists of one parental strand and one new strand. DNA Replication Is Semiconservative

4. Origin of replication 5’ 3’ 5’ Frame of reference

5. Origin of replication 5’ 3’ 5’ Frame of reference DnaA proteins ( ) – initiate replication

6. Origin of replication DnaA proteins ( ) – initiate replication 5’ 3’ 5’ Replication bubble – binding generates a “bubble” allowing for bidirection replication

7. Helicase enzyme ( ) – unwinds the double helix 5’ 3’ 5’ – replication bubble expands Replication forks (expanding in opposite directions)

8. Helicase enzyme ( ) – unwinds the double helix 5’ 3’ 5’ – replication bubble expands Single-strand binding proteins ( ) – keep the strands separated

9. Helicase enzyme ( ) – unwinds the double helix 5’ 3’ 5’ – replication bubble expands Single-strand binding proteins ( ) – keep the strands separated Gyrase ( ) – relieves supercoiling ahead of replication fork

10. Primase enzyme ( ) – builds a short RNA segment 5’ 3’ 5’

11. Primase enzyme ( ) – builds a short RNA segment 5’ 3’ 5’ – RNA serves as a “primer” ( )

12. 5’ 3’ 5’

13. Primase enzyme ( ) – builds a short RNA segment 5’ 3’ 5’

14. 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand Primase enzyme ( ) – builds a short RNA segment – RNA serves as a “primer” ( )

15. DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group 5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand

16. 5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

17. 5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

18. 5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

19. LE 16-13 New strand 5 end Phosphate Base Sugar Template strand 3 end 5 end 3 end 5 end 3 end 5 end 3 end Nucleoside triphosphate DNA polymerase Pyrophosphate

20. = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

21. = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ Primase enzyme ( ) – builds a short RNA segment

22. = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ Primase enzyme ( ) – builds a short RNA segment – RNA serves as a “primer” ( ) When DNA Polymerase III encounters a road block, it disengages and starts building from a newly generated primer

23. = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

24. = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

25. = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Leading strand synthesis always heads toward its respective replication fork Lagging strand synthesis always heads away from its respective replication fork

26. Process of leading strand synthesis is called “continuous” Process of lagging strand synthesis is called “discontinuous” = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Leading strand synthesis always heads toward its respective replication fork Lagging strand synthesis always heads away from its respective replication fork

27. Disconnected segments resulting from discontinuous synthesis are called Okazaki fragments = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Okazaki framents

28. RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’

29. RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ DNA Polymerase I ( ) – uses neighboring fragment’s free 3’ hydroxyl to extend and displace RNA nucleotides

30. RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ DNA Polymerase I ( ) – uses neighboring fragment’s free 3’ hydroxyl to extend and displace RNA nucleotides 5’

31. RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ DNA Polymerase I ( ) – uses neighboring fragment’s free 3’ hydroxyl to extend and displace RNA nucleotides 5’

32. = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ DNA Ligase ( ) – joins disconnected neighboring fragments

33. = RNA primer for leading strand = RNA primer for lagging strand 3’ 5’ 3’ 5’ DNA Ligase ( ) – joins disconnected neighboring fragments – yields one contiguous piece of newly synthesized complementary DNA