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REPLICATION

REPLICATION. Copying DNA. 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

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REPLICATION

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  1. REPLICATION Copying DNA

  2. 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 A complex reaction © 2010 Paul Billiet ODWS

  3. Image Credit: DNA 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 DNAImage Credit: 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 ODWS

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

  7. The cell cycle Some cells may stay in this stage for over a year Cytokinesis division of the cytoplasm G0 M First growth phase. Varies in length Mitosis G1 G2 Interphase Copying of chromosomes = replication Second growth period S G1 + S + G2 = INTERPHASE © 2010 Paul Billiet ODWS

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

  9. Meselson and Stahl’s results DNA LightMediumHeavy 0 0.3 0.7 1.0 1.1 1.5 1.9 2.5 3.0 4.1 0 +1.9 0 + 4.1 Controls GENERATIONS © 2010 Paul Billiet ODWS

  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 bandsA strong light band and a weaker medium • This carries on, the light band getting stronger. © 2010 Paul Billiet ODWS

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

  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 ODWS

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

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

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

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

  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 ODWS

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

  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 ODWS

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

  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 ODWS

  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 ODWS

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

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