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LE 16-7

The mechanism of DNA Replication. LE 16-7. 5  end. Hydrogen bond. 3  end. 1 nm. 3.4 nm. 3  end. 0.34 nm. 5  end. Key features of DNA structure. Partial chemical structure. Space-filling model. When during the cell cycle is DNA synthesized? Draw.

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LE 16-7

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  1. The mechanism of DNA Replication LE 16-7 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end Key features of DNA structure Partial chemical structure Space-filling model When during the cell cycle is DNA synthesized? Draw

  2. The Basic Principle: Base Pairing • Each strand acts as a template for building a new strand in replication • Parent dsDNA molecule unwinds & base pairs are broken - two new daughter strands built based on base-pairing rules Draw

  3. LE 16-9_1 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

  4. A simple model of DNA replication LE 16-9_4 The first step is separation of the two parental DNA strands. The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. Synthesis of complementary strands Predicted by Watson and Crick Semiconservative model of DNA replication

  5. First replication Second replication Parent cell Various proposed models of DNA replication Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. LE 16-10 Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple-mentary strand. Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

  6. Meselson and Stahl experimentally supported one of the replication models How & which one?

  7. Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N Heavy radioisotope Light radioisotope LE 16-11 Why label nitrogen? Less dense DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) More dense Second replication First replication Conservative model Supported by data Semiconservative model Dispersive model

  8. Replication begins • at origin of replication (ori) • Creation of replication bubble with replication forks at each end (Draw) • Hundreds to thousands of oris on eukaryotic chromosome • Usually one on bacterial chromosome • Proceeds in both directions from each origin, until the entire molecule is copied

  9. Parental (template) strand 0.25 µm Origin of replication Daughter (new) strand LE 16-12 Replication fork Bubble Two daughter DNA molecules In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). Arrowheads mark replication forks. In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome.

  10. Elongating a New DNA Strand • Basic components Template DNA DNA polymerase DNA precursors deoxynucleotide triphosphates (dATP, dCTP, dGTP,dTTP)

  11. New strand Template strand 5¢ end 3¢ end 5¢ end 3¢ end LE 16-13 Sugar Base Phosphate DNA polymerase 3¢ end 5’ 3¢ end Pyrophosphate Nucleoside triphosphate 5¢ end 5¢ end

  12. Specificity of DNA polymerase • only adds nucleotides to the free 3hydroxyl end of dsDNA • New DNA strand made only in 5’-3’direction Draw

  13. 3¢ 5¢ primer Parental DNA Leading strand 5¢ LE 16-14 3¢ Okazaki fragments Lagging strand 3¢ 5¢ DNA pol III Template strand Leading strand Lagging strand Template strand DNA ligase Overall direction of replication

  14. LE 16-16 Overall direction of replication Lagging strand Leading strand Origin of replication Leading strand Lagging strand OVERVIEW DNA pol III Leading strand DNA ligase Replication fork 5¢ DNA pol I 3¢ Primase Lagging strand Parental DNA DNA pol III Primer 3¢ 5¢

  15. Other components of the DNA replication machinery? DNA helicase- to unwind DNA Single strand binding proteins- to stabilize ssDNA DNA ligase- to seal gap in sugar-phosphate backbone (make phosphodiester bond) between Okazaki fragments

  16. A Closer Look at Lagging Strand Synthesis Primase joins RNA nucleotides into a primer. 3¢ 5¢ 3¢ 5¢ Template strand LE 16-15_1 Overall direction of replication

  17. Primase joins RNA nucleotides into a primer. 3¢ 5¢ 3¢ 5¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. LE 16-15_2 3¢ 5¢ RNA primer 3¢ 5¢ Overall direction of replication

  18. Primase joins RNA nucleotides into a primer. 3¢ 5¢ 3¢ 5¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. LE 16-15_3 3¢ 5¢ RNA primer 3¢ 5¢ After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3¢ 3¢ 5¢ 5¢ Overall direction of replication

  19. Primase joins RNA nucleotides into a primer. 3¢ 5¢ 3¢ 5¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. LE 16-15_4 3¢ 5¢ RNA primer 3¢ 5¢ After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3¢ 3¢ 5¢ 5¢ After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5¢ 3¢ 3¢ 5¢ Overall direction of replication

  20. Primase joins RNA nucleotides into a primer. 3¢ 5¢ 3¢ 5¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. LE 16-15_5 3¢ 5¢ RNA primer 3¢ 5¢ After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3¢ 3¢ 5¢ 5¢ After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5¢ 3¢ 3¢ 5¢ DNA pol I replaces the RNA with DNA, adding to the 3¢ end of fragment 2. 5¢ 3¢ 3¢ 5¢ Overall direction of replication

  21. Primase joins RNA nucleotides into a primer. 3¢ 5¢ 3¢ 5¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. LE 16-15_6 3¢ 5¢ RNA primer 3¢ 5¢ After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3¢ 3¢ 5¢ 5¢ After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5¢ 3¢ 3¢ 5¢ DNA pol I replaces the RNA with DNA, adding to the 3¢ end of fragment 2. 5¢ 3¢ 3¢ 5¢ DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. The lagging strand in the region is now complete. 5¢ 3¢ 3¢ 5¢ Overall direction of replication

  22. Animation: Lagging Strand

  23. Animation: DNA Replication Review

  24. Proofreading and Repairing DNA • DNA polymerases proofread • Replace mismatched nt in new DNA • Also • Mismatch repair: repair enzymes correct errors in base pairing 2. Nucleotide excision repair: enzymes cut out and replace damaged stretches of DNA

  25. Example DNA exposure to ultraviolet (UV) light induces chemical crosslinks between adjacent thymines (thymine dimers) How to repair?

  26. A thymine dimer distorts the DNA molecule. LE 16-17 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease Repair synthesis by a DNA polymerase fills in the missing nucleotides. DNA polymerase DNA ligase DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete.

  27. Is DNA replication of linear chromosomes ever complete? Consider the tips (ends) of the leading and lagging strands.

  28. 5¢ Leading strand End of parental DNA strands Lagging strand 3¢ LE 16-18 Last fragment Previous fragment RNA primer Lagging strand 5¢ 3¢ Primer removed but cannot be replaced with DNA because no 3¢ end available for DNA polymerase Removal of primers and replacement with DNA where a 3¢ end is available 5¢ 3¢ Second round of replication 5¢ New leading strand 3¢ New leading strand 5¢ 3¢ Further rounds of replication Shorter and shorter daughter molecules

  29. Ends of eukaryotic chromosomes • Tipped with many copies of a short DNA repeat called telomeres (e.g. human telomere sequence TTAGGG x 100-1,000) • Added by telomerase , a ribozyme (made of RNA and proteins) Function:Telomeres postpone loss of important genes near ends after each cell division. Is telomerase found in all eukaryotic cells? NO, mostly in germ cells but NOT in somatic cells.

  30. What will happen to DNA in cells that continually divide such as epithelial cells (skin, gut)? Make a prediction about the length of chromosomes in skin cells from a 80 year old versus a 4 year old. Cancer cells are characterized in part by their continuous cell division. Shouldn’t they ultimately die from loss of genes due to shortening of chromosomes? Hypothesize why they continue to divide without injury? Cancer cells express telomerase, which prevents chromosome shortening

  31. LE 16-19 Labelled telomeres Questions? 1 µm

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