04 dna replication-v2

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04 dna replication-v2

  1. 2. DNA Polymerase <ul><li>DNA polymerase – enzyme which synthesizes nucleotide chains </li></ul><ul><ul><li>in prokaryotes: DNA polymerase I, II, III, IV & V </li></ul></ul><ul><ul><li>in eukaryotes: over 15 different types </li></ul></ul><ul><li>nucleotide chains only form in the 5’  3’ direction </li></ul><ul><li>DNA polymerase III is primarily responsible for DNA replication in prokaryotes </li></ul>
  2. 3. DNA Polymerase III Substrates <ul><li>DNA template </li></ul><ul><li>deoxyribonucleoside triphosphates (dNTPs) </li></ul><ul><li>RNA primer </li></ul>5’ 3’ 5’ 3’ 5’ 3’
  3. 4. Step 1 <ul><li>Primase makes a short RNA primer on the exposed single-stranded DNA ( ssDNA ). </li></ul>5’ 3’ 5’ 3’
  4. 5. DNA Replication: Elongation <ul><li>Nucleotides are added to the 3’ end </li></ul><ul><li>Nucleotide = Nucleoside triphoshate = nitrogen base + deoxyribose + triphosphate </li></ul><ul><li>As each nucleotide is added, the last two phosphate groups are hydrolyzed to form pyrophosphate. </li></ul><ul><li>Pyrophosphate is broken down into two phosphates </li></ul>
  5. 6. <ul><li>As each nucleotide is added, the last two phosphate groups are hydrolyzed to form pyrophosphate. </li></ul><ul><ul><li>The exergonic hydrolysis of pyrophosphate to two inorganic phosphate molecules drives the polymerization of the nucleotide to the new strand. </li></ul></ul>Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 16.11
  6. 7. Steps 2, 3, and 4 <ul><li>DNA polymerase III binds to the end of the RNA primer. </li></ul><ul><li>The appropriate nucleoside triphosphate binds to the polymerase. </li></ul><ul><li>A pyrophosphate group (PPi) is cleaved, while the nucleotide is added to the end of the nucleotide chain. </li></ul>A A T TTP P P
  7. 8. Pyrophosphate <ul><li>http://bcs.whfreeman.com/thelifewire/pages/bcs-main.asp?s=00020&n=01000&i=01020.01&v=category&o=|00510|00570|00520|00530|00540|00550|00580|00PRS|00560|00010|00020|00030|00040|00050|00060|00070|00120|00080|00090|00130|00100|00110|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|24000|25000|26000|27000|28000|29000|30000|31000|32000|33000|34000|35000|36000|37000|38000|39000|40000|41000|42000|43000|44000|45000|46000|47000|48000|49000|50000|51000|52000|53000|54000|55000|56000|57000|58000|99000|&ns=0&uid=0&rau=0 </li></ul>
  8. 9. DNA Polymerase <ul><li>DNA polymerase III catalyzes the elongation of DNA molecules by adding nucleotides to the 3’ end of a pre-existing strand. </li></ul><ul><li>DNA polymerase I replaces the RNA primer with DNA complementary to the template </li></ul>
  9. 10. Priming DNA synthesis with RNA Fig. 16.13
  10. 11. DNAP III: elongates DNA strand DNAP I: replaces RNA with DNA Fig. 16.14 Video: http://highered.mcgraw-hill.com/olc/dl/120076/bio23.swf
  11. 12. 5’ 5’ 3’ 3’ helicase SSBPs gyrase primase DNA polymerase III Direction of Replication 5’ 3’
  12. 13. Okazaki fragment Direction of Replication
  13. 14. Overview Fig. 16.16 http://kvhs.nbed.nb.ca/gallant/biology/replication_overview.jpg
  14. 15. Connecting Lagging Strands <ul><li>DNA polymerase I – removes the RNA primer </li></ul><ul><ul><li>occurs in the 5’  3’ direction </li></ul></ul><ul><li>DNA ligase – connects the sugar-phosphate backbone of Okazaki fragments </li></ul><ul><ul><li>Okazaki fragments are typically 1000 to 2000 nucleotides (NTs) in length </li></ul></ul>
  15. 16. <ul><li>The lagging strand requires formation of a new primer as the replication fork progresses. </li></ul><ul><li>After the primer is formed, DNA polymerase can add new nucleotides away from the fork until it runs into the previous Okazaki fragment. </li></ul><ul><li>The primers are converted to DNA before DNA ligase joins the fragments together. </li></ul>Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
  16. 17. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 16.15
  17. 18. Tutorials <ul><li>http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/dna-rna2.swf </li></ul><ul><li>http://www.wiley.com/legacy/college/boyer/0470003790/animations/replication/replication.swf </li></ul><ul><li>http://www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf </li></ul><ul><li>http://www.johnkyrk.com/DNAreplication.html </li></ul>
  18. 19. <ul><li>Mistakes during the initial pairing of template nucleotides and complementary nucleotides occurs at a rate of one error per 10,000 base pairs. </li></ul><ul><li>DNA polymerase proofreads each new nucleotide against the template nucleotide as soon as it is added. </li></ul><ul><li>If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis. </li></ul><ul><li>The final error rate is only one per billion nucleotides. </li></ul>Enzymes proofread DNA during its replication and repair damage in existing DNA Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
  19. 20. Replication Animation <ul><li>Replication Animation </li></ul>
  20. 21. <ul><li>Limitations in the DNA polymerase create problems for the linear DNA of eukaryotic chromosomes. </li></ul><ul><li>The usual replication machinery provides no way to complete the 5’ ends of daughter DNA strands. </li></ul><ul><ul><li>Repeated rounds of replication produce shorter and shorter DNA molecules. </li></ul></ul>The ends of DNA molecules are replicated by a special mechanism Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
  21. 22. Telomeres <ul><li>Telomeres are found at the ends of linear chromosomes. </li></ul><ul><li>No genes are located in this region. </li></ul><ul><li>Repetitive sequence of TTAGGG; between 100x to 1000x repetition. </li></ul>
  22. 23. <ul><li>The ends of eukaryotic chromosomal DNA molecules, the telomeres , have special nucleotide sequences. </li></ul><ul><ul><li>In human telomeres, this sequence is typically TTAGGG, repeated between 100 and 1,000 times. </li></ul></ul><ul><li>Telomeres protect genes from being eroded through multiple rounds of DNA replication. </li></ul>Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 16.19a
  23. 25. Telomerase <ul><li>Each round of replication shortens the telomeres. </li></ul><ul><li>Shortened telomere length is thought to be a signal to start the process of aging. </li></ul><ul><li>Telomerase is an enzyme that restores the length of telomeres. </li></ul>
  24. 26. <ul><li>Eukaryotic cells have evolved a mechanism to restore shortened telomeres. </li></ul><ul><li>Telomerase uses a short molecule of RNA as a template to extend the 3’ end of the telomere. </li></ul><ul><ul><li>There is now room for primase and DNA polymerase to extend the 5’ end. </li></ul></ul><ul><ul><li>It does not repair the 3’-end “overhang,” but it does lengthen the telomere. </li></ul></ul>Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 16.19b
  25. 28. <ul><li>Telomerase is not present in most cells of multicellular organisms. </li></ul><ul><li>Therefore, the DNA of dividing somatic cells tend to become shorter. </li></ul><ul><li>Thus, telomere length may be a limiting factor in the life span of certain tissues and the organism. </li></ul><ul><li>Telomerase is present in sex cells, ensuring that zygotes have long telomeres. </li></ul><ul><li>Active telomerase is also found in cancerous somatic cells. </li></ul><ul><ul><li>This overcomes the progressive shortening that would eventually lead to self-destruction of the cancer. </li></ul></ul>Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
  26. 29. Replication Overview - 1 <ul><li>helicase unwinds the double stranded DNA structure creating a replication fork </li></ul><ul><li>the single stranded region of the replication fork are maintained by SSBPs </li></ul><ul><li>gyrase relieves the tension ahead of the replication fork </li></ul>
  27. 30. Replication Overview - 2 <ul><li>two original parent strands serve as templates for the new daughter strands </li></ul><ul><li>daughter strands are produced in one of two methods </li></ul><ul><ul><li>leading strand (continuous polymerization) </li></ul></ul><ul><ul><li>lagging strand (discrete polymerization) </li></ul></ul><ul><ul><ul><li>1000 – 2000 NT Okazaki fragments joined together </li></ul></ul></ul>
  28. 31. Replication Overview - 3 <ul><li>primase begins each new daughter strand with a short RNA primer </li></ul><ul><li>DNA polymerase III extends a DNA strand from the RNA primer </li></ul><ul><li>DNA polymerase I removes the RNA primer AND fills it in with DNA </li></ul><ul><li>DNA ligase joins the sugar-phosphate backbones of all adjacent DNA segments </li></ul>
  29. 32. Mistakes in DNA Replication <ul><li>less than 1 error in 10 7 NTs </li></ul><ul><li>exonuclease – enzymes which can cut out sections of nucleic acids </li></ul><ul><li>DNA polymerase I and III have polymerase and exonuclease activity to fix mistakes </li></ul>

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