1. REPLICATION Copying DNA

2. A complex reaction Uncoiling of parent molecule Unzipping the two sister strands to reveal the sequence of bases Reading the sequence of bases Choosing the complementary nucleotide building blocks Lining up the nucleotides and bonding them together Checking for errors Recoiling the two DNA molecules. All controlled by enzymes in particular DNA polymerase © 2010 Paul Billiet ODWSODWS

3. Image Credit: DNA polymerase IIIDNA polymerase III

4. A very rapid reaction The average length the DNA molecule in a bacteriophage (a large virus) is 34µm long 100 000 base pairs 10 000 turns (10 base pairs per revolution) Replication time 2 minutes Replication speed 83 revolutions per second. Phage particle releasing its DNA Image Credit: http://www.biochem.wisc.edu/http://www.biochem.wisc.edu/

5. Multiple replication forks Eukaryotes have much more DNA They have their DNA divided up into many molecules (chromosomes) Replication in eukaryotes begins at many points along each chromosome This reduces the time taken. © 2010 Paul Billiet ODWSODWS

6. Where and when does replication occur? In the nucleus of eukaryotes During interphase During S-phase © 2010 Paul Billiet ODWSODWS

7. The cell cycle G 1 + S + G 2 = INTERPHASE First growth phase. Varies in length Copying of chromosomes = replication Some cells may stay in this stage for over a year Second growth period M G1G1 S G2G2 G0G0 Cytokinesis division of the cytoplasm © 2010 Paul Billiet ODWSODWS

8. Meselson & Stahl’s experiment ultracentrifuge DNA settles a level because of its density Samples taken at timed intervals And DNA extracted Bacteria fed on N-15 labelled food for several generations Bacteria switched to N-14 labelled food © 2010 Paul Billiet ODWSODWS

9. Meselson and Stahl’s results 00.30.71.01.11.51.92.53.04.1 0 +1.90 + 4.1 GENERATIONS Controls Light Medium Heavy DNA © 2010 Paul Billiet ODWSODWS

10. Observations Initially all the DNA is “heavy” Only one band appears After one generation there is one band but it is “medium” After two generations there are two equal bands “Medium” and “Light” After three generations there are two bands A strong light band and a weaker medium This carries on, the light band getting stronger. © 2010 Paul Billiet ODWSODWS

11. Interpretation of the results GENERATION 1 2 3 0 © 2010 Paul Billiet ODWSODWS

12. Interpretation At each generation the DNA molecule splits A new strand is fabricated alongside the old one The is semi-conservative replication. © 2010 Paul Billiet ODWSODWS

13. E.coli caught in the act! 2 strands of parental DNA Initiation point Growing point Newly formed daughter strands 1962 autoradiograph by John Cairns of a replicating E. coli chromosome

14. A = T T = A C  G G  C C  G T = A A = T T = A C  G A = T T = A T = T A = T A G T A T A = T T = A C  G G  C C  G T = A A = T T C A T A Helicase Untwisting the helix & breaking the hydrogen bonds © 2010 Paul Billiet ODWSODWS

15. T C A T A G T A T A = T T = A C  G G  C C  G T = A A = T A T DNA Polymerase III Deoxynucleoside triphosphates Complementary base pairing Adding in the nucleotides © 2010 Paul Billiet ODWSODWS

16. A = T T = A C  G G  C C  G T = A A = T T = A C  G A = T T = A T = T A = T T = A C  G G  C C  G T = A A = T T = A C  G A = T T = A T = T A = T Two daughter strands © 2010 Paul Billiet ODWSODWS

17. Added complications DNA helicase III cannot start the process of replication A small primer of RNA is needed first This requires another enzyme RNA primase. © 2010 Paul Billiet ODWSODWS

18. T C A T A G T A T A = T T = A C  G G  C C  G T = A A = T DNA Polymerase III G T A T RNA primase © 2010 Paul Billiet ODWSODWS

19. Added complications DNA polymerase III can only add nucleotides on one way (5’ to 3’) BUT the DNA molecule is antiparallel One strand can be replicated directly as it unzips (the leading strand) The other strand needs to wait until a certain amount is unzipped (the lagging strand). © 2010 Paul Billiet ODWSODWS

20. 3’ 5’ 3’ 5’ Leading strand Lagging strand Okazaki fragments 5’ 3’ 5’ 3’ © 2010 Paul Billiet ODWSODWS

21. Added complications The lagging strand is replicated in fragments about 1000 base pairs long OKAZAKI fragments Each fragment starts with an RNA primer. © 2010 Paul Billiet ODWSODWS

22. Added complications At the end the RNA primers are removed by another enzyme, DNA polymerase I Replaces the primers with DNA nucleotides The ends of the Okazaki fragments are stuck together using DNA ligase. © 2010 Paul Billiet ODWSODWS

23. DNA polymerase I replaces the RNA primers with DNA Ligase connects the fragments Gaps need connecting © 2010 Paul Billiet ODWSODWS