1. DNA replication

2. SBI4U0- DNA Replication
Cells need to have the ability to reproduce for many reasons.
When cells make copies of themselves, they must ensure that their entire genetic code is present in both the old and new cell
DNA must replicate to ensure 2 complete copies of the genetic code. This is why cells must undergo the process of MITOSIS (remember, sister chromatids are copies of the same chromosome!)

3. DNA Replication
During cell division, the genetic material has to be divided up between daughter cells or gametes. (Mitosis - production of daughter cells; Meiosis- production of gametes)
DNA replicates first in INTERPHASE

4. Remember This?
The Cell Cycle

5. The Cell Cycle Two Main Stages Interphase Cell Division
Growth 1, Synthesis, Growth 2
Cell Division
Mitosis, Cytokinesis

6. THE CELL CYCLE G1 G2 S Telophase Anaphase Cytokinesis Metaphase
Prophase G1 G2 S

7. INTERPHASE Period between cell divisions
Cell undergoes growth, replication of DNA, obtaining energy, making hormones, repairing damage, fighting disease
*Most of the cell’s time is spent in this phase – length varies depending on the organism and cell type

8. Prophase

9. Metaphase

10. Anaphase

11. Telophase

12. Back to Interphase
S Phase
How does DNA replicate?

13. THE CELL CYCLE G1 G2 S Telophase Anaphase Cytokinesis Metaphase
Prophase G1 G2 S

14. DNA Replication
Produces two identical copies of the chromosome during S phase of interphase
Catalyzed by many enzymes

15. DNA Structure and composition:
DNA strands are antiparallel, meaning they run in opposite directions
One strand runs in the 5'  3' direction, the other in the 3'  5' direction

16. DNA Structure and composition:
The 3' end of one strand ends with the –OH on C3 (3rd carbon of the sugar molecule)
The 5' end of a strand ends with a phosphate group attached to the C5 (5th carbon of the pentose sugar)

18. DNA Replication
Watson and Crick’s model of DNA structure suggested how DNA could replicate
Hydrogen bonds break
Helix unzip
Each strand act as a template to build new strand
At this time there were no experimental results to support this hypothesis

19. 3 models of replication:
Semiconservative replication would produce molecules with both old and new DNA, but each molecule would be composed of one old strand and one new one.
Conservative replication would leave intact the original DNA molecule and generate a completely new molecule.
Dispersive replication would produce two DNA molecules with sections of both old and new DNA interspersed along each strand.

20. Semiconservative Model
DNA begins by unzipping itself  the hydrogen bonds break between strands
2 strands separate, and each acts as a template or guide for directing the synthesis of the new strand.
"Semiconservative" is used to describe this process since each new DNA molecule contains 1 old strand and 1 new strand
This model was proven by Meselson & Stahl

21. Meselson and Stahl Experiment
Meselson and Stahl concluded that DNA replication is not conservative but semiconservative
Each strand acts as template for the building of the complementary strand
Each DNA strand is composed of one parent strand and one newly synthesized strand

22. Meselson-Stahl experiment
E. coli bacteria was grown using 15N, a denser isotope of N, for several generations.
When 15N DNA was centrifuged in cesium chloride, which forms a density gradient, all the DNA settled in one single layer, forming a band.

23. M and S then switched to 14N for one generation.
If DNA replication was conservative, they should have seen two bands, one containing the old DNA with 15N, the other containing the new DNA with 14N.
If DNA replication was semiconservative, they should have seen one intermediate band.

24. After two generations with 14N, they saw two bands:
One intermediate band (mix of 15N and 14N)
One lighter band (only 14N)
Over several generations, they found that the intermediate band did not go away even after they stopped using 15N.
This indicates that the original 15N DNA was still there, still acting as a template.

25. Meselson-Stahl experiment

27. Video Overview
and Stahl Experiment

28. DNA Replication

29. Steps Separating the DNA strands Building Complementary Strands
Linking of Nitrogenous Bases and Proof-reading

30. 1) Separating the DNA strands
The replication of DNA must take place in many steps. The entire DNA strand cannot be replicated at once because the unraveled DNA would be too large for the cell.
The piece of unwould DNA that is being replicated creates a DNA Replication Bubble
At each end of the bubble is the Replication Fork
The enzyme helicase breaks the hydrogen bonds holding the two complementary parent strands together, resulting in an unwound, unzipped helix that terminates at the replication fork.
The enzyme gyrase / topoisomerase relieves any tension from the unwinding of the double helix

31. 1. Separating DNA Strands
Initiated at sites along DNA called Replication Origins – made of specific nucleotide sequences
Enzymes and other proteins work together to unwind and stabilize the double helix

32. The Players: DNA helicase DNA gyrase (aka topoisomerase)
SSBs (single-strand binding proteins)

33. DNA Helicase
Recognizes specific nucleotide sequence (origin of replication)
Unwinds double helix by breaking H bonds between complementary base pairs
Opens up one or more replication bubbles

35. DNA Gyrase / Topoisomerase
Relieves the tension produced by the unwinding of DNA
Cuts one or two of the strands near the replication fork so that they can untangle and rejoin
Topoisomerase in action:

36. Single-stranded Binding Proteins
SSBs
Keep separated DNA strands apart by blocking hydrogen bonding
Keep the templates (single DNA strands) straight

37. DNA Replication – Step 3
Single-stranded binding proteins (SSB’s) anneal to the newly exposed template strands, preventing them from reannealing by blocking hydrogen bonding.

38. 1. DNA unwinding and unzipping
1. The enzyme helicase breaks the H-bonds holding the two complementary parent strands together, resulting in an unzipped helix that terminates at the replication fork.
2. The enzyme gyrase relieves any tension from the unwinding of the double helix.
3. Single-stranded binding proteins (SSBs) anneal to the newly exposed template strands, preventing them from re-annealing.

39. DNA Replication - Overview

40. 2) Elongation: Building of Complementary Strands
Replication begins in two directions from the origin
Toward direction of replication fork on one strand
Away from direction of replication fork on the other
DNA polymerase builds new strands
DNA polymerase III used in prokaryotes
DNA polymerase III can only synthesize DNA in the 5’ to 3’ direction

41. 2. Building complementary strands
Because there may be more than one origin of replication in eukaryotes, more than one replication fork may exist
Link-replication animation:

42. The Players
Primase
DNA polymerase
DNA ligase

43. Primase
DNA nucleotides can only be added by DNA polymerase to an existing strand.
Primase builds RNA primers which are used to initiate DNA replication

44. DNA Replication
DNA polymerase III cannot initiate a new strand by itself so a RNA primer is required
The enzyme primase lays down RNA primers that will be used by DNA polymerase III as a starting point to build the new complementary strands.

45. DNA polymerase III
Takes free nucleotides found within the cell and adds them in the 5’ to 3’ direction, first to the RNA primer and then to the DNA nucleotide that was just added
The parent strand is used as a template

46. Leading (good guy) strand
The daughter strand that grows continuously towards the replication fork as the double helix unwinds
Occurs quickly
Requires only a single RNA primer at replication origin

47. Leading Strand
DNA polymerase III adds the appropriate deoxyribonucleoside triphosphates to the 3 prime end of the new strand using the template strand as a guide.
The energy in the phosphate bonds is used to drive the process. The leading strand is built continuously toward the replication fork.

48. Lagging (scumbag) strand
The 3’ to 5’ parent strand is a problem for DNA polymerase since it must synthesize in the 5’ to 3’ direction!

49. Lagging strand
Built in short segments (in the 5’ to 3’ direction) away from the replication fork
Requires many RNA primers

50. Lagging Strand
A lagging strand composed of short segments of DNA, known as Okazaki fragments, is built discontinuously away from the replication fork.

51. DNA polymerase I DNA ligase
Removes the RNA primers once they have been used and replaces them with the appropriate DNA sequence
DNA ligase
Joins the Okazaki fragments into one strand by the creation of phosphodiester bonds

52. Replacing the RNA Primers
DNA polymerase I excises the RNA primers and replaces them with the appropriate deoxyribonucleotides. DNA ligase joins the gaps in the Okazaki fragments by the creation of a phosphodiester bond.

53. Link-leading vs lagging animation: http://highered. mcgraw- hill
Link-leading vs lagging animation: hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::53 5::/sites/dl/free/ /120076/micro04.swf

55. 3) Quality Control
DNA polymerase III acts as a proof- reader by checking the newly synthesized strand for any incorrectly inserted bases
If a mistake is found, it backs up and replaces the incorrect base with the correct one

56. DNA Replication – Final Step!
Errors missed by DNA polymerase III is “proofread” and repaired by DNA polymerase II by excising incorrectly paired nucleotides at the end of the complementary strand and adding the correct nucleotides.
Similar repair mechanisms also help to repair the damage caused by carcinogens (toxic chemicals, UV light and other radiation)

57. Termination
The process of DNA replication ends when all the replication bubbles meet. The DNA molecules rewind to gain their normal helical form. There is a problem though, due to the removal of primers- a little bit of a gap always remains on the lagging strand
Therefore, entire chromosome can never be fully copied!
It is estimated that with each round of duplication, around 100 bps of DNA are lost
To deal with this problem, each chromosome has a series of bases the essentially code for nothing tagged onto the end of the chromosomes. These buffer ‘ends’ are called Telomeres. Courtesy of the enzyme ‘telomerase’
Slow erosion of the telomeres (after approximately 50 replications) leads to the aging and death of the cell.

61. Animations DNA Replication Fork:
How Nucleotides are Added:

62. DNA Replication Simulator:

63. Telomeres, Aging and Cancer
It is interesting to note that older people have shorter telomeres than younger people at the end of their chromosomes.
Certain types of cells have no apparent shortage to their telomeres ex. Sperm and cancer cells. Follow the links below to gather more information on telomeres: