DNA Replication Repair

1. DNA Replication & Repair Biology 11 Chapter 14

2. What is the Heredity Material? Chromosome existence had been known since the late 1800�s Mendel completed extensive work on heredity The nature of Mendel�s heredity factors remained a mystery until 1940�s

3. DNA vs. Protein It was also known that chromosomes were a compilation of DNA and proteins So what was this �transmissible� factor that was inherited from generation to generation DNA Protein The prevailing theory of the day was that the transmissible factor was protein

4. Why Protein Proteins have almost a limitless variation in structure and function With all of the different proteins it was reasonable to assume that a significant level of biological diversity could be reached DNA on the other hand was a simple chemical structure (the actual secondary structure was not known until 1950�s) Only four bases (nucleotides) A, T, G & C

5. DNA as the Hereditary Material The next slides will outline a series of classical experiments which conclusively showed that DNA and not proteins is the hereditary material Frederick Griffith � 1928s Oswald Avery � 1940s Hershey � Chase � 1940s Watson � Crick � 1950s

6. Griffith In 1928 Griffith reported a mysterious phenomenon Transformation At the time Griffith was working on a vaccine for Streptococcus pneumoniae Griffith was working with two strains R � rough strain avirulent S � smooth strain virulent

7. Griffith

8. Griffith�s Experiment

9. Avery�s Experiment Began with a normal S type cell Exposed to heat and other treatments to yield an extract of only DNA, RNA, & Protein

10. Avery�s Experiment

11. Critics of Avery�s Experiment Even after Avery�s experiment most scientist still believed that protein was the transforming factor These scientists claimed that the enzymatic treatments were not sufficient to remove all of the proteins More studies were performed with viruses

12. Hershey � Chase They wanted to conclusively answer the question of whether the transforming factor was DNA or protein Used the T2 bacteriophage virus and E. coli What is a bacteriophage? What is a virus? All viruses are acellular entities which are made up of a protein coat which surrounds the genome Some viruses can have an envelope (cell membrane) around the protein coat

13. Bacteriophage A bacteriophage is a �bacteria eating� virus Many types of these viruses exist Inject viral DNA into host cells and cause the host cell to make many copies of the virus

14. Bacteriophage Life Cycle The life cycle is as follows Docking Adsorption Replication It is important to remember that the virus injects only DNA and that it then uses the cellular machinery of the bacterial cell to make more copies of its genome and the proteins More copies of viral genome More copies of viral proteins Assembly Release

16. Other Viruses It uses its genetic information (DNA or RNA) and directs a cell to make more copies of the virus HIV Herpes Hepatitis Small Pox HPV

17. Hershey-Chase Are �genes� made of DNA or protein Used a T2 virus that only infects E. coli cells What was known T2 injects �genes� into bacteria T2 is made up of only DNA & protein

18. Hershey-Chase Using radioactive labels these researches tagged either the DNA or protein population P32 Why does this tag only DNA S35 Why does this tag only proteins

19. Hershey & Chase

20. Hershey & Chase

22. Hershey & Chase Conclusion The genetic material that redirects the functions of an E. coli cell is DNA After these results were published proponents of the protein hypothesis had to admit that DNA, not protein, must be the hereditary material

23. Watson & Crick In the 1950s Watson and Crick proposed their model for DNA secondary structure The structure provided �rules� or directions for how to copy itself provided one of the strands was acting as a template

24. Meselson � Stahl Experiment When replication occurs during S phase is the new DNA � old and � new Or do the parent strands serve as templates and then reanneal to one another Conservative vs. Semi conservation Replication

25. Meselson � Stahl Experiment To answer this question Meselson and Stahl exploited the differences between N14 and N15 N15 is a heavy isotope with one extra neutron The difference in mass creates a difference in density This difference in density can be seen when centrifugation techniques are employed

28. Mechanisms For Replication The mechanism for DNA replication are complex and have only been known for the last 25 years The key discovery insight began with the discovery of DNA polymerase DNA polymerases Catalyze the formation of the daughter strand There are many types of polymerases enzymes These enzymes only work in the 5� to 3� direction

29. Initiation of DNA replication Step 1 � opening the helix Proteins bind to specific DNA sequences known as origins of replication Bacteria have one Eukaryotes have thousands AT rich regions � Why? Helicases aid in the opening of the helix

30. Initiation of DNA replication Role of SSBP Single stranded binding proteins After the helix has opened it is prevented from re-annealing by the action of these proteins These proteins stabilize single stranded DNA

31. Initiation of DNA replication Step 2 � binding of RNA primers Primase adds short stretches of RNA primers Purpose is to give DNA polymerase a 3�OH group from which to add new DNA nucleotides Two primers are put down as the replication bubble opens

32. Replication Elongation Step 3 After the primers are in place an enzyme known as DNA polymerase III (bacteria; eukaryotes has a similar polymerase but it has a different name) will add new nucleotides to the daughter strand as directed by the template strand Replication must proceed in the 5� to 3� direction

33. DNA Polymerase

34. Chemistry of DNA Polymerase

38. Replicating Linear Chromosomal Ends What is the chance that primase would add a primer exactly on the last base of the lagging strand? NOT LIKELY

40. How To Correct Lagging Strand End Shortening Linear chromosome have ends known as telomeres Telomeres do not contain genes that code for proteins Telomers are stretches of repetitive bases repeated over and over again TTAGGG in humans repeated thousands of times

41. How To Correct Lagging Strand End Shortening Telomerase is an enzyme synthesizes DNA from an RNA template contained in the active site of the enzyme Telomerase binds to the ends of linear DNA as adds these repetitive bases Telomerase then lengthen the overhang on the lagging strand

42. Telomerase

43. Telomerase Complications Telomerase is not active in most somatic cells This raises an interesting questions If telomerase is not active then it is reasonable to assume that cells must be in G0 and not growing and dividing If telomerase is active then would it lead to uncontrolled cell growth and cancer

44. Telomerase Complications Conflicting results Knockout mice which do not have telomerase develop tumors Suggests that telomerase activity is not necessary for tumor formation But many cancerous cells in humans have telomerase activity which allows then to continue to grow and divide Adding active telomerase to cultured cells allows them to grow past a �normal� life span

45. Risks To DNA Replication Synthesizing DNA is risky business As it turns out DNA polymerase can make mistakes in matching complementary DNA What happens if during replication DNA mismatches, such as G=T were not corrected? Mutation would occur at such a rapid rate life itself would not be possible

46. Risks To DNA Replication DNA polymerase inserts the incorrect base once in every 100,000 bases Error rate of 1 x 10-5 At this rate your genome would be riddle with mutations But as it turns out DNA polymerase can proofread

47. Proofreading DNA polymerase contains a subunit which is capable of excising a nucleotide in the 3� to 5� direction Known as a 3� to 5� exonuclease This reduces the error to one mistake per 10 million bases Error rate of 1 x 10-7

48. Proofreading

49. Proofreading Mechanism Explains 5� to 3� Replication Requirement In terms of chemistry an incoming 3�OH could just as easily attack a 5� triphosphate group So why then did all DNA replication evolve to synthesize DNA in the 5� to 3� direction?

50. Proofreading Is this error rate of 10-7 acceptable? NO!! During or shortly after DNA replication a system known as mismatch repair is active The enzymes involved in mismatch repair were discovered using E. coli as the model system MutS, MutL, MutH & others Humans have MLH, MSH etc. Humans with defective MMR genes are genetically predisposed to HPNCC

51. Mismatch Repair in E. Coli

52. DNA Damage Even after DNA is synthesized and proofread and mismatches repaired, all is not well DNA is under constant assault Spontaneous DNA damage Breathing Spontaneous deamination of C Physical DNA damage UV light and radiation Chemical DNA damage Benzo a pyrene Nitrates and nitrites Aflatoxin B1 moldy peanuts and corn

53. Spontaneous DNA Damage Oxidative Damage Aerobic Respiration causes DNA damage everyday Reactive oxygen radicals are created from ETC Deamination Reactions � C become U

54. Deamination of Cytosine Spontaneous non-enzymatic deamination of cytosine occurs at a rate of 10-7 cytosine per day Equal to 100 cytosine to uracil conversions each day Based on this information why does DNA contain thymine and not uracil?

55. C to U Transversions

56. Physical Damage Ionizing radiation leads to DNA double strand breaks Cause deletions of large sections of DNA Gene amplification events Translocation events � part of one chromosome attaches to another chromosome UV light causes a specific type of mutation

57. Repairof Thymine Dimers

58. Xeroderma Pigmentosum Rare autosomal recessive disease in humans Extreme sensitivity to UV light Skin develops lesions even after short exposure

59. Chemical Damage Alkalization of DNA is common when exposed to certain chemicals Alkalization interferes with proper base pairing and could lead to increased mistakes during replication Damaged bases can be repaired using BER

60. DNA Repair All cells have repair systems in place to deal with DNA damage Mismatch repair - MMR Base excision repair - BER Nucleotide excision repair - NER Direct repair Others