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DNA Replication

DNA Replication. Double helix structure of DNA. “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick. Directionality of DNA. You need to number the carbons! it matters!.

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DNA Replication

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  1. DNA Replication

  2. Double helix structure of DNA “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

  3. Directionality of DNA • You need to number the carbons! • it matters! nucleotide PO4 N base 5 CH2 This will beIMPORTANT!! O 1 4 ribose 3 2 OH

  4. 5 The DNA backbone PO4 • Putting the DNA backbone together • refer to the 3 and 5 ends of the DNA • the last trailing carbon base CH2 5 O 4 1 C 3 2 O P –O O Sounds trivial, but…this will beIMPORTANT!! O base CH2 5 O 4 1 2 3 OH 3

  5. Anti-parallel strands • Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons • DNA molecule has “direction” • complementary strand runs in opposite direction THIS WILL CAUSE A PROBLEM FOR REPLICATION 5 3 3 5

  6. hydrogen bonds covalent phosphodiester bonds Bonding in DNA 5 3 3 5 ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA?

  7. Copying DNA • Replication of DNA • base pairing allows each strand to serve as a template for a new strand • new strand is 1/2 parent template & 1/2 new DNA

  8. Let’s meetthe team… DNA Replication • Large team of enzymes coordinates replication

  9. Replication: 1st step • Unwind DNA • helicase enzyme • unwinds part of DNA helix • stabilized by single-stranded binding proteins • PREVENTS DNA MOLECULE FROM CLOSING! • DNA gyrase • Enzyme that prevents tangling upstream from the replication fork gyrase helicase single-stranded binding proteins replication fork

  10. Replication: 2nd step • Add RNA primer • DNA BY RNA Primase • Why must this be done? • DNA can’t be added to an existing strand of nucleotides

  11. Replication: 3rd step • Build daughter DNA strand • add new complementary bases • With the help of the enzyme DNA polymerase III But… We’re missing something! What? Where’s theENERGYfor the bonding! DNA Polymerase III

  12. Energy of Replication Where does energy for bonding usually come from? We comewith our ownenergy! energy YourememberATP!Are there other waysto get energyout of it? energy Are thereother energynucleotides?You bet! And weleave behind anucleotide! CTP ATP TTP GTP AMP ADP GMP TMP CMP modified nucleotide

  13. Energy of Replication • The nucleotides arrive as nucleosides • DNA bases with P–P–P • P-P-P = energy for bonding • DNA bases arrive with their own energy source for bonding: by breaking off two phosphate groups • bonded by enzyme: DNA polymerase III ATP GTP TTP CTP

  14. 4th step • Replacement of RNA primer by DNA • Done by DNA polymerase I

  15. Before we solve the Problem. Lets review DNA replication

  16. Limits of DNA polymerase III • DNA polyermase III can only add nucleotides to an existing strand • DNA polymerase III can only add nucleotides to 3 end of a DNA strand • WHY IS THAT A PROBLEM?

  17. Okazaki ligase 3 3 3 3 3 3 3 5 5 5 5 5 5 5 Leading & Lagging strands Limits of DNA polymerase III • can only build onto 3 end of an existing DNA strand  Okazaki fragments Lagging strand growing replication fork  Leading strand Lagging strand • Okazaki fragments • joined by ligase • “spot welder” enzyme DNA polymerase III Leading strand • continuous synthesis

  18. ENTER MR. OKAZAKI

  19. 3 3 3 3 3 3 DNA polymerase III 5 5 5 5 5 5 DNA replication on the lagging strand RNA primer is added • built by primase • serves as starter sequence for DNA polymerase III HOWEVER short segments called Okazaki fragments are made because it can only go in a 5 3 direction growing replication fork primase RNA

  20. ligase 3 3 3 3 5 5 5 5 Replacing RNA primers with DNA NEXT DNA polymerase I • removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I growing replication fork RNA STRANDS ARE GLUED TOGETHER BY DNA LIGASE

  21. Lagging strand DNA replication review

  22. 3 3 3 3 5 5 5 5 Houston, we have a problem! Chromosome erosion All DNA polymerases can only add to 3 end of an existing DNA strand DNA polymerase I growing replication fork DNA polymerase III RNA Loss of bases at 5 endsin every replication • chromosomes get shorter with each replication • limit to number of cell divisions?

  23. 3 3 3 3 5 5 5 5 Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap • limit to ~50 cell divisions growing replication fork telomerase Telomerase • enzyme extends telomeres • can add DNA bases at 5 end • different level of activity in different cells • high in stem cells & cancers -- Why? TTAAGGG TTAAGGG TTAAGGG

  24. direction of replication Replication fork DNA polymerase III lagging strand DNA polymerase I 3’ primase Okazaki fragments 5’ 5’ ligase SSB 3’ 5’ 3’ helicase DNA polymerase III 5’ leading strand 3’ SSB = single-stranded binding proteins

  25. Roger Kornberg 2006 Arthur Kornberg 1959 DNA polymerases • DNA polymerase III • 1000 bases/second! • main DNA builder • DNA polymerase I • 20 bases/second • editing, repair & primer removal DNA polymerase III enzyme

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