1. DNA E. McIntyre IB Biology HL

2. DNA is the Genetic Material Therefore it must Replicate faithfully. Have the coding capacity to generate proteins and other products for all cellular functions. “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.” -Francis Crick

3. Watson & Crick in action

4. Models for DNA replication 1) Semiconservative model: Daughter DNA molecules contain one parental strand and one newly replicated strand. 2) Conservative model: Parent strands transfer information to an intermediate, then the intermediate gets copied. The parent helix is conserved, the daughter helix is completely new. 3) Dispersive model: Parent helix is broken into fragments, dispersed, copied then assembled into two new helices. New and old DNA are completely Dispersed.

5. (a) Hypothesis 1: Semi-conservative replication (b) Hypothesis 2: Conservative replication Intermediate molecule (c) Hypothesis 3: Dispersive replication MODELS OF DNA REPLICATION

6. Meselson and Stahl Semi-conservative replication of DNA

8. The families of nitrogenous bases

9. DNA Replication Since DNA replication is semiconservative, therefore the helix must be unwound. John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short. DNA replication is semiconservative. Each strand of both replication forks is being copied. DNA replication is bidirectional. Bidirectional replication involves two replication forks, which move in opposite directions

10. Evidence points to bidirectional replication Label at both replication forks

11. The Enzymes of Replication

12. A 3’ hydroxyl group is necessary for addition of nucleotides

13. DNA Polymerase contains a Proofreading subunit Accuracy of DNA polymerases is essential. -Error rate is less than 1 in 10 8

14. Proofreading by DNA polymerase

15. DNA Exonuclease

16. DNA replication is semi-discontinuous Continuous synthesis Discontinuous synthesis

17. Proteins & the Replication Fork

18. Protein complexes of the replication fork: DNA polymerase DNA primase DNA Helicase ssDNA binding protein Sliding Clamp Clamp Loader DNA Ligase DNA Topoisomerase

19. DNA primase synthesizes an RNA primer to initiate DNA synthesis on the lagging strand

20. Replication of the Lagging Strand

21. DNA ligase seals nicks left by lagging strand replication

22. DNA helicase unwinds the DNA duplex ahead of DNA polymerase creating single stranded DNA that can be used as a template

23. DNA helicase moves along one strand of the DNA

24. ssDNA binding proteins are required to “iron out” the unwound DNA

25. ssDNA binding proteins bind to the sugar phosphate backbone leaving the bases exposed for DNA polymerase

26. DNA polymerase is not very processive (ie it falls off the DNA easily). A “sliding clamp” is required to keep DNA polymerase on and allow duplication of long stretches of DNA

27. A “clamp loader:” complex is required to get the clamp onto the DNA

28. Lagging strand synthesis

30. The supercoiling ahead of the fork needs to be relieved or tension would build up (like coiling as spring) and block fork progression.

31. Type I topoisomerases: Make nicks in one DNA strands Can relieve supercoiling Type II topoisomersases: Make nicks in both DNA strands (double strand break) Can relieve supercoiling and untangle linked DNA helices Both types of enzyme form covalent intermediates with the DNA Supercoiling is relieved by the action of Topoisomerases

32. Topoisomerase I Action

33. Topoisomerase II Action

35. Because dividing cells require greater topoisomerase activitydue to increased DNA synthesis, topoisomerase inhibitors are used as chemotherapeutic agents. e.g. Camptothecin -- Topo I inhibitor Doxorubicin -- Topo II inhibitor These drugs act by stablilzing the DNA-Topoisomerase complex. Also, some antibiotics are inhibitors of the bacterial- specific toposisomerase DNA gyrase e.g. ciprofloxacin Topoisomerases as drug targets

36. DNA is replicated during S phase of the Cell Cycle

37. In S phase, DNA replication begins at origins of replication that are spread out across the chromosome

38. Each origin of replicaton initiates the formation of bidirectional replication forks

39. Errors lead to over replication of specific chromosomal regions. (= gene amplification) This seen commonly in cancer cells and can be an important prognostic indicator. It can also contribute to acquired drug resistance. Origins of replication are strictly controlled so that they “fire” only once per cell cycle

40. Errors of DNA Replication and Disease The rate of misincorporation of bases by DNA polymerase is extremely low, however repeated sequences can cause problems In particular, trinucleotide repeats cause difficulties which can lead to expansion of these sequences. Depending where the repeat is located expansion of the sequence can have severe effects on the expression of a gene or the function of a protein.

41. Several inherited diseases are associated with expansion of trinucleotide repeat sequences.

42. Have no fear…it’s efficient!! Check out these stats… Polymerase III It’s fast: up to 1,000 dNTPs added/sec/enzyme It’s highly processive: >500,000 dNTPs added before dissociating It’s accurate: makes 1 error in 10 7 dNTPs added, with proofreading, this gives a final error rate of 1 in 10 10 overall.