1. DNA Replication Section 4.3 Page 217 Why do we need to replicate our DNA? When does DNA replication occur in a cell?

2. Background Cell division: mitosis + cytokinesis DNA replicated in interphase, prior to mitosis Each daughter cell must have an exact copy of the parent cell’s DNA

3. But how does replication occur? Scientists in the 50s had 3 proposed models: 1. Semi-conservative 2. Conservative 3. Dispersive But which one is right???

4. Semi-conservative – Two parental strands separate and each serves as a template for a new progeny strand. Newly-synthesized DNA molecules has one old strand, and one new strand DNA REPLICATION: Models Semi- conservative ConservativeDispersive

5. Conservative – the two parental strands stay together, and somehow produce another daughter helix with completely new strands. Newly-synthesized DNA molecules has two new strands. DNA REPLICATION: Models Semi-conservativeConservativeDispersive

6. Dispersive – DNA becomes fragmented so that new and old DNA coexist in the same strand after replication. Newly-synthesized DNA molecule has pieces of old and new strands interspersed. DNA REPLICATION: Models Semi-conservativeConservativeDispersive

8. Meselson-Stahl experiment, 1958 Purpose: to elucidate the mode of replication Experimental model: E. coli

9. Grew E. coli in a medium enriched with a heavy nitrogen isotope ( 15 N) ◦ Denser-than-normal DNA Switched cells to ordinary medium with 14 N, and allowed DNA replication  Will 14 N be incorporated into DNA strands with 15 N?

10. Centrifuged the DNA within a density gradient: Separates components according to density *A centrifuge is a device that spins a solution at high speeds, the spinning splits up the different components in a mixture based on density http://highered.mcgraw- hill.com/olc/dl/120076/bio22.swf

11. Possible results:

13. Observed: First generation – One intermediate band Second generation – One light/one intermediate Conclusion: DNA replication is semi-conservative

14. DNA REPLICATION: THE DETAILS

15. Three stages: Stage 1: Initiation ◦ DNA strands are separated ◦ A small portion of RNA is annealed to the exposed strands to “prime” them for replication Stage 2: Elongation: ◦ DNA polymerase III builds a new strand of DNA by incorporating nucleotides Stage 3: Termination

16. Stage 1: Initiation Separation of strands: DNA strands are “unzipped” by DNA helicase ◦ Hydrogen bonds between complementary bases are broken

17. Single-stranded binding proteins (SSBs) bind to exposed strands to prevent re-annealing of strands. DNA gyrase relieves torsion tension by cutting and re-annealing the two strands

18. Priming: DNA polymerase cannot start incorporating nucleotides on its own ◦ Needs an existing 3’ end of a nucleic acid A short segment of RNA (a “primer” – 10 to 60 nucleotides long) provides that 3’ end

19. RNA Primase synthesizes the primer and anneals it to the template strand. DNA polymerase can then add on DNA nucleotides

20. Stage 2: Elongation New strand is synthesized in the 5’ to 3’ direction (added on to the end with the -OH group) Catalyzed by DNA polymerase III

21. Free bases are floating in the nucleoplasm as deoxyribonucleoside triphosphates. Energy required for DNA synthesis is provided by hydrolyzing the bond between the 1 st and 2 nd phosphates

22. Characteristics of elongation: A. Bi-directionality B. Semi-discontinuity

23. A. Elongation is bi-directional Elongation proceeds in two directions, outwards from the origin of replication. The junction where the strands are still joined is called the replication fork.

24. DNA synthesis occurs simultaneously using both strands as templates ◦ A replication bubble forms between two replication forks

25. B. Elongation is semi-discontinuous DNA synthesis always occurs in the 5’ to 3’ direction (of the new strand!) The two template strands are antiparallel  Only one strand can be built continuously http://highered.mcgraw- hill.com/olc/dl/120076/micro04.swf

26. Leading strand – Uses the 3’ to 5’ template strand as its guide ◦ Is built continuously, towards the replication fork

27. Lagging strand – Uses the 5’ to 3’ template strand as its guide ◦ Is built discontinuously in short fragments RNA primase constantly adds new RNA primers along the template strand. The fragments are called Okazaki fragments. = site of new primer

28. Removal of the RNA primers, and joining of the Okazaki fragments:

29. EnzymeRole DNA polymerase Iremoves the RNA primers; replaces them with the proper deoxyribonucleosides DNA ligasejoins the fragments together (phosphodiester bonds) Removal of the RNA primers, and joining of the Okazaki fragments:

30. Stage 3: Termination Two replication forks meet each other; or DNA Polymerase III reaches the end of a strand

31. Problem: Shortening of telomeres Telomeres: The ends of DNA. Contain repetitive sequences. Protects the chromosome from degradation. Loss of telomeric DNA occurs on the lagging strand with each replication. http://spine.rutgers.edu/cellbio/assets/flash/tel.h tm

32. Lagging strand: No free 3’ end to replace RNA with DNA 

33. Approximately 50 replications before the telomeres become too short. Telomere shortening linked to aging.

34. Telomerase Telomerase - enzyme that prevents shortening of telomeres Present in cells that need to divide constantly: white blood cells, germ line cells May be present in cancerous cells

35. Proofreading DNA polymerase III and DNA polymerase I are constantly proofreading the progeny strand as it is synthesized. Both have exonuclease activity  can identify incorrectly added nucleotides, backtrack, and excise them (cut them out) before continuing synthesis.

37. Further proofreading mechanisms are in place when synthesis is completed.

38. Recap: Three important properties of DNA replication: 1. DNA replication is semi-conservative 2. DNA replication is bi-directional 3. DNA replication is semi-discontinuous …recall what these mean!!

39. Enzymes/proteins involved in DNA replication: DNA helicase DNA gyrase single-stranded binding proteins RNA primase DNA polymerase III DNA polymerase I DNA ligase DNA telomerase