1. Replication RNA Synthesis Decoding the Genetic Code Noel Murphy

2. Reference Sources Hartl & Jones, Genetics: Analysis of Genes and Genomes, 6 th Edition Chapter 6 – Replication Chapter 10 – Transcription and the code Klug & Cummings, Essentials of Genetics 5 th Edition Chapter 11 – Replication Chapter 13 – Transcription and the code Lectures http://www.tcd.ie/Genetics/staff/Noel_Murphy.htm

3. DNA is the Genetic Material Therefore it must (1)Replicate faithfully. (2)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

4. Replication

5. The Dawn of Molecular Biology April 25, 1953 Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

6. 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

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

8. Testing Models for DNA replication Matthew Meselson and Franklin Stahl (1958)

9. Meselson and Stahl Semi-conservative replication of DNA Isotopes of nitrogen (non-radioactive) were used in this experiment

10. Generations 0 0.3 0.7 1.0 1.1 1.5 1.9 2.5 3.0 4.1 0 and 1.0 mixed 0 and 4.1 mixed HH HL LL + HL HH HL LL LH Equilibrium Density Gradient Centrifugation Detection of semiconservative replication in E. coli by density-gradient centrifugation. The position of a band of DNA depends on its content of 14 N amd 15 N. After 1.0 generation, all the DNA molecules are hybrids containing equal amounts of 14 N and 15 N

11. DNA replication Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTPs)

12. Replication as a process Double-stranded DNA unwinds. The junction of the unwound molecules is a replication fork. A new strand is formed by pairing complementary bases with the old strand. Two molecules are made. Each has one new and one old DNA strand.

13. 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.

14. Replication can be Uni- or Bidirectional

15. John Cairns Grow cells for several generations Small amounts of 3 H thymidine are incorporated into new DNA Grow for brief period of time Add a high concentration of 3 H- thymidine in media with low concentration of 3 H- thymidine Bacterial culture *T Dense label at the replication fork where new DNA is being made *T All DNA is lightly labeled with radioactivity *T Cairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.

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

17. Features of DNA Replication 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

18. Arthur Kornberg (1957) Protein extracts from E. coli + Template DNA Is new DNA synthesized?? - dNTPs (substrates) all 4 at once - Mg 2+ (cofactor) - ATP (energy source) - free 3OH end (primer) In vitro assay for DNA synthesis Used the assay to purify a DNA polymerizing enzyme DNA polymerase I

19. 3 Kornberg also used the in vitro assay to characterize the DNA polymerizing activity - dNTPs are ONLY added to the 3 end of newly replicating DNA -therefore DNA synthesis occurs only in the 5 to 3 direction 33 5 3 5 5 3 5 5 3 5 5 3 5 3 Parental template strand New progeny strand

20. THIS LEADS TO A CONCEPTUAL PROBLEM Consider one replication fork: 5 3 5 3 Direction of unwinding Continuous replication 5 3 Primer 5 3 5 3 Discontinuous replication

21. Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968)

22. Evidence for Semi-Discontinuous Replication (pulse-chase experiment) Bacteria are replicating Bacterial culture Add 3 H Thymidine For a SHORT time (i.e. seconds) Flood with non-radioactive T Allow replication To continue Harvest the bacteria at different times after the chase Isolate their DNA Separate the strands (using alkali conditions) Run on a sizing gradient smallest largest Radioactivity will only be in the DNA that was made during the pulse

23. smallest largest Results of pulse-chase experiment Pulse 5 3 5 3 Direction of unwinding 3 5 Primer 5 3 5 3 * * * * * * Chase

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

25. Features of DNA Replication DNA replication is semiconservative –Each strand of template DNA is being copied. DNA replication is bidirectional –Bidirectional replication involves two replication forks, which move in opposite directions DNA replication is semidiscontinuous –The leading strand copies continuously –The lagging strand copies in segments (Okazaki fragments) which must be joined

26. The Enzymology In 1957, Arthur Kornberg demonstrated the existence of a DNA polymerase - DNA polymerase I DNA Polymerase I has THREE different enzymatic activities in a single polypeptide: a 5 to 3 DNA polymerizing activity a 3 to 5 exonuclease activity a 5 to 3 exonuclease activity of DNA Replication

27. Subsequent hydrolysis of PPi drives the reaction forward Nucleotides are added at the 3'-end of the strand The 5 to 3 DNA polymerizing activity

28. Why the exonuclease activities? The 3'-5' exonuclease activity serves a proofreading function It removes incorrectly matched bases, so that the polymerase can try again.

29. Proof reading activity of the 3 to 5 exonuclease. DNAPI stalls if the incorrect ntd is added - it cant add the next ntd in the chain Proof reading activity is slow compared to polymerizing activity, but the stalling of DNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizing activity and remove the incorrect base.

30. How? 1) Base-pairing specificity at the active site - correct geometry in the active site occurs only with correctly paired bases BUT the wrong base still gets inserted 1/ 10 4 -10 5 dNTPs added 2) Proofreading activity by 3-5 exonuclease - removes mispaired dNTPs from 3 end of DNA - increases the accuracy of replication 10 2 -10 3 fold 3) Mismatch repair system - corrects mismatches AFTER DNA replication DNA Replication is Accurate (In E. coli: 1 error/10 9 -10 10 dNTPs added)

31. Is DNA Polymerase I the principal replication enzyme?? In 1969 John Cairns and Paula deLucia isolated a mutant bacterial strain with only 1% DNAP I activity (polA) - mutant was super sensitive to UV radiation - but otherwise the mutant was fine i.e. it could divide, so obviously it can replicate its DNA Conclusion: DNAP I is NOT the principal replication enzyme in E. coli

32. - DNAP I is too slow (600 dNTPs added/minute – would take 100 hrs to replicate genome instead of 40 minutes) - DNAP I is only moderately processive (processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template) Conclusion: There must be additional DNA polymerases. Biochemists purified them from the polA mutant Other clues….

33. - functions in multiple processes that require only short lengths of DNA synthesis - has a major role in DNA repair (Cairns- deLucia mutant was UV-sensitive) - its role in DNA replication is to remove primers and fill in the gaps left behind - for this it needs the nick-translation activity So if its not the chief replication enzyme then what does DNAP I do?

34. A total of 5 different DNAPs have been reported in E. coli DNAP I: functions in repair and replication DNAP II: functions in DNA repair (proven in 1999) DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in 1999) DNAP V: functions in DNA repair (discovered in 1999) The DNA Polymerase Family

35. The "real" replicative polymerase in E. coli Its fast: up to 1,000 dNTPs added/sec/enzyme Its highly processive: >500,000 dNTPs added before dissociating Its accurate: makes 1 error in 10 7 dNTPs added, with proofreading, this gives a final error rate of 1 in 10 10 overall. DNA Polymerase III