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2007-2008 Synthesis of DNA June.21.2010
DNA synthesis occurs by the process of  replication. During replication, each of the two parental strands of  DNA serves a...
Each molecule generated by the  replication process contains one intact parental  strand and  one newly  synthesized stran...
In eukaryotes, DNA replication occurs during the  S phase  of the cell cycle The cell divides during the next phase ( M ),...
Cultured MDCK cells Day 1 Day 3
Watson and Crick 1953 article in Nature
Double helix structure of DNA “ It has not escaped our notice that the specific pairing we have postulated immediately sug...
Directionality of DNA <ul><li>You need to number the carbons! </li></ul><ul><ul><li>it matters! </li></ul></ul>OH CH 2 O 4...
The DNA backbone <ul><li>Putting the DNA backbone together </li></ul><ul><ul><li>refer to the 3   and 5   ends of the DN...
Anti-parallel strands <ul><li>Nucleotides in DNA backbone are bonded from phosphate to sugar between 3   & 5   carbons <...
Bonding in DNA … . strong  or  weak  bonds? How do the bonds fit the mechanism for copying DNA?  3  5  3  5  covalent ...
Base pairing in DNA <ul><li>Purines </li></ul><ul><ul><li>adenine (A) </li></ul></ul><ul><ul><li>guanine (G) </li></ul></u...
Copying DNA <ul><li>Replication of DNA </li></ul><ul><ul><li>base pairing allows each strand to serve as a  template  for ...
DNA Replication  <ul><li>Large team of enzymes coordinates replication </li></ul>Let ’ s meet the team …
Replication: 1st step <ul><li>Unwind DNA </li></ul><ul><ul><li>helicase  enzyme </li></ul></ul><ul><ul><ul><li>unwinds par...
Replication: 2nd step DNA Polymerase III But … We ’ re missing  something ! What? Where ’ s the ENERGY for the bonding ! <...
Energy of Replication <ul><li>Where does energy for bonding  usually  come from? </li></ul>energy ATP GTP TTP CTP ADP AMP ...
Energy of Replication <ul><li>The nucleotides arrive as  nucleosides </li></ul><ul><ul><li>DNA bases with  P – P – P </li>...
<ul><li>Adding bases  </li></ul><ul><ul><li>can only add nucleotides to  3   end  of a  growing  DNA strand </li></ul></u...
energy 3  5  5  5  3  need “primer” bases to add on to energy energy energy 3  no energy to bond energy energy energ...
Leading & Lagging strands <ul><li>Limits of DNA polymerase III </li></ul><ul><ul><li>can only build onto 3   end of an ex...
Replication fork / Replication bubble leading strand lagging strand leading strand lagging strand leading strand lagging s...
Starting DNA synthesis: RNA primers <ul><li>RNA primer </li></ul><ul><ul><li>built by  primase </li></ul></ul><ul><ul><li>...
Replacing RNA primers with DNA <ul><li>DNA polymerase I </li></ul><ul><ul><li>removes sections of RNA primer and replaces ...
Chromosome erosion <ul><li>Loss of bases at 5   ends   in every replication </li></ul><ul><ul><li>chromosomes get shorter...
Telomeres <ul><li>Repeating, non-coding sequences at the end of chromosomes = protective cap </li></ul><ul><ul><li>limit t...
Replication fork 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ helicase SSB = single-stranded binding proteins primase DNA  polymerase III DNA  ...
DNA polymerases <ul><li>DNA polymerase III </li></ul><ul><ul><li>1000 bases/second ! </li></ul></ul><ul><ul><li>main  DNA ...
Editing & proofreading DNA <ul><li>1000 bases/second =  lots of typos! </li></ul><ul><li>DNA polymerase I   </li></ul><ul>...
Fast & accurate! <ul><li>It takes  E .  coli  <1 hour to copy  5 million base pairs in its single chromosome  </li></ul><u...
What does it really look like? 1 2 3 4
2007-2008 Any Questions??
Energy of Replication <ul><li>Where does energy for bonding  usually  come from? </li></ul>energy ATP GTP TTP ATP ADP AMP ...
<ul><li>Adding bases  </li></ul><ul><ul><li>can only add nucleotides to  3   end  of the  growing DNA strand </li></ul></...
5  3  3  5  3  5  3  5  no energy to bond 
energy 5  3  3  5  3  5  3  5  ligase
Chromosome erosion <ul><li>Loss of bases at 5   ends   in every replication </li></ul><ul><ul><li>chromosomes get shorter...
Replication fork 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ direction of replication
DNA synthesis in prokaryotes <ul><li>Replication is bidirectional. </li></ul><ul><li>Replication is semi-conservative. </l...
Bidirectional replication of a circular chromosome Replication begins at the point of origin ( oriC ) and  proceeds in bot...
Unwinding of Parental Strands Topoisomerases:   can break phosphodiester bonds and rejoin them relieve the supercoiling of...
以上です。
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Biochem synthesis of dna

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Biochem synthesis of dna

  1. 1. 2007-2008 Synthesis of DNA June.21.2010
  2. 2. DNA synthesis occurs by the process of replication. During replication, each of the two parental strands of DNA serves as a template for the synthesis of a Complementary strand.
  3. 3. Each molecule generated by the replication process contains one intact parental strand and one newly synthesized strand .
  4. 4. In eukaryotes, DNA replication occurs during the S phase of the cell cycle The cell divides during the next phase ( M ), and each daughter cell receives an exact copy of the DNA of the parent cells.
  5. 5. Cultured MDCK cells Day 1 Day 3
  6. 6. Watson and Crick 1953 article in Nature
  7. 7. 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
  8. 8. Directionality of DNA <ul><li>You need to number the carbons! </li></ul><ul><ul><li>it matters! </li></ul></ul>OH CH 2 O 4  5  3  2  1  PO 4 N base ribose nucleotide This will be IMPORTANT !!
  9. 9. The DNA backbone <ul><li>Putting the DNA backbone together </li></ul><ul><ul><li>refer to the 3  and 5  ends of the DNA </li></ul></ul><ul><ul><ul><li>the last trailing carbon </li></ul></ul></ul>OH O 3  PO 4 base CH 2 O base O P O C O – O CH 2 1  2  4  5  1  2  3  3  4  5  5  Sounds trivial, but … this will be IMPORTANT !!
  10. 10. Anti-parallel strands <ul><li>Nucleotides in DNA backbone are bonded from phosphate to sugar between 3  & 5  carbons </li></ul><ul><ul><li>DNA molecule has “direction” </li></ul></ul><ul><ul><li>complementary strand runs in opposite direction </li></ul></ul>3  5  5  3 
  11. 11. Bonding in DNA … . strong or weak bonds? How do the bonds fit the mechanism for copying DNA? 3  5  3  5  covalent phosphodiester bonds hydrogen bonds
  12. 12. Base pairing in DNA <ul><li>Purines </li></ul><ul><ul><li>adenine (A) </li></ul></ul><ul><ul><li>guanine (G) </li></ul></ul><ul><li>Pyrimidines </li></ul><ul><ul><li>thymine (T) </li></ul></ul><ul><ul><li>cytosine (C) </li></ul></ul><ul><li>Pairing </li></ul><ul><ul><li>A : T </li></ul></ul><ul><ul><ul><li>2 bonds </li></ul></ul></ul><ul><ul><li>C : G </li></ul></ul><ul><ul><ul><li>3 bonds </li></ul></ul></ul>
  13. 13. Copying DNA <ul><li>Replication of DNA </li></ul><ul><ul><li>base pairing allows each strand to serve as a template for a new strand </li></ul></ul><ul><ul><li>new strand is 1/2 parent template & 1/2 new DNA </li></ul></ul>
  14. 14. DNA Replication <ul><li>Large team of enzymes coordinates replication </li></ul>Let ’ s meet the team …
  15. 15. Replication: 1st step <ul><li>Unwind DNA </li></ul><ul><ul><li>helicase enzyme </li></ul></ul><ul><ul><ul><li>unwinds part of DNA helix </li></ul></ul></ul><ul><ul><ul><li>stabilized by single-stranded binding proteins </li></ul></ul></ul>single-stranded binding proteins replication fork helicase
  16. 16. Replication: 2nd step DNA Polymerase III But … We ’ re missing something ! What? Where ’ s the ENERGY for the bonding ! <ul><li>Build daughter DNA strand </li></ul><ul><ul><li>add new complementary bases </li></ul></ul><ul><ul><li>DNA polymerase III </li></ul></ul>
  17. 17. Energy of Replication <ul><li>Where does energy for bonding usually come from? </li></ul>energy ATP GTP TTP CTP ADP AMP GMP TMP CMP modified nucleotide energy We come with our own energy ! And we leave behind a nucleotide ! You remember ATP ! Are there other ways to get energy out of it? Are there other energy nucleotides? You bet !
  18. 18. Energy of Replication <ul><li>The nucleotides arrive as nucleosides </li></ul><ul><ul><li>DNA bases with P – P – P </li></ul></ul><ul><ul><ul><li>P-P-P = energy for bonding </li></ul></ul></ul><ul><ul><li>DNA bases arrive with their own energy source for bonding </li></ul></ul><ul><ul><li>bonded by enzyme: DNA polymerase III </li></ul></ul>ATP GTP TTP CTP
  19. 19. <ul><li>Adding bases </li></ul><ul><ul><li>can only add nucleotides to 3  end of a growing DNA strand </li></ul></ul><ul><ul><ul><li>need a “starter” nucleotide to bond to </li></ul></ul></ul><ul><ul><li>strand only grows 5  3  </li></ul></ul>Replication DNA Polymerase III DNA Polymerase III DNA Polymerase III DNA Polymerase III energy energy energy energy 3  3  5  B.Y.O. ENERGY ! The energy rules the process 5 
  20. 20. energy 3  5  5  5  3  need “primer” bases to add on to energy energy energy 3  no energy to bond energy energy energy 3  5   ligase
  21. 21. Leading & Lagging strands <ul><li>Limits of DNA polymerase III </li></ul><ul><ul><li>can only build onto 3  end of an existing DNA strand </li></ul></ul>Leading strand Lagging strand Okazaki fragments <ul><li>Leading strand </li></ul><ul><ul><li>continuous synthesis </li></ul></ul><ul><li>Lagging strand </li></ul><ul><ul><li>Okazaki fragments </li></ul></ul><ul><ul><li>joined by ligase </li></ul></ul><ul><ul><ul><li>“ spot welder” enzyme </li></ul></ul></ul>DNA polymerase III   growing replication fork 5  5  5  5  3  3  3  5  3  5  3  3  ligase Okazaki 3  5 
  22. 22. Replication fork / Replication bubble leading strand lagging strand leading strand lagging strand leading strand lagging strand DNA polymerase III 5  3  5  3  5  3  3  5  5  3  5  3  5  3  5  3  growing replication fork growing replication fork 5  5  5  5  5  3  3  5  5  5  3 
  23. 23. Starting DNA synthesis: RNA primers <ul><li>RNA primer </li></ul><ul><ul><li>built by primase </li></ul></ul><ul><ul><li>serves as starter sequence for DNA polymerase III </li></ul></ul><ul><li>Limits of DNA polymerase III </li></ul><ul><ul><li>can only build onto 3  end of an existing DNA strand </li></ul></ul>growing replication fork primase RNA DNA polymerase III 5  5  5  3  3  3  5  3  5  3  5  3 
  24. 24. Replacing RNA primers with DNA <ul><li>DNA polymerase I </li></ul><ul><ul><li>removes sections of RNA primer and replaces with DNA nucleotides </li></ul></ul>But DNA polymerase I still can only build onto 3  end of an existing DNA strand growing replication fork DNA polymerase I RNA 5  5  5  5  3  3  3  3  ligase
  25. 25. Chromosome erosion <ul><li>Loss of bases at 5  ends in every replication </li></ul><ul><ul><li>chromosomes get shorter with each replication </li></ul></ul><ul><ul><li>limit to number of cell divisions? </li></ul></ul>DNA polymerase III All DNA polymerases can only add to 3  end of an existing DNA strand growing replication fork DNA polymerase I RNA Houston, we have a problem ! 5  5  5  5  3  3  3  3 
  26. 26. Telomeres <ul><li>Repeating, non-coding sequences at the end of chromosomes = protective cap </li></ul><ul><ul><li>limit to ~50 cell divisions </li></ul></ul><ul><li>Telomerase </li></ul><ul><ul><li>enzyme extends telomeres </li></ul></ul><ul><ul><li>can add DNA bases at 5  end </li></ul></ul><ul><ul><li>different level of activity in different cells </li></ul></ul><ul><ul><ul><li>high in stem cells & cancers -- Why? </li></ul></ul></ul>telomerase growing replication fork TTAAGGG TTAAGGG TTAAGGG 5  5  5  5  3  3  3  3 
  27. 27. Replication fork 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ helicase SSB = single-stranded binding proteins primase DNA polymerase III DNA polymerase III DNA polymerase I ligase Okazaki fragments leading strand lagging strand SSB direction of replication
  28. 28. DNA polymerases <ul><li>DNA polymerase III </li></ul><ul><ul><li>1000 bases/second ! </li></ul></ul><ul><ul><li>main DNA builder </li></ul></ul><ul><li>DNA polymerase I </li></ul><ul><ul><li>20 bases/second </li></ul></ul><ul><ul><li>editing, repair & primer removal </li></ul></ul>DNA polymerase III enzyme Arthur Kornberg 1959 Roger Kornberg 2006
  29. 29. Editing & proofreading DNA <ul><li>1000 bases/second = lots of typos! </li></ul><ul><li>DNA polymerase I </li></ul><ul><ul><li>proofreads & corrects typos </li></ul></ul><ul><ul><li>repairs mismatched bases </li></ul></ul><ul><ul><li>removes abnormal bases </li></ul></ul><ul><ul><ul><li>repairs damage throughout life </li></ul></ul></ul><ul><ul><li>reduces error rate from 1 in 10,000 to 1 in 100 million bases </li></ul></ul>
  30. 30. Fast & accurate! <ul><li>It takes E . coli <1 hour to copy 5 million base pairs in its single chromosome </li></ul><ul><ul><li>divide to form 2 identical daughter cells </li></ul></ul><ul><li>Human cell copies its 6 billion bases & divide into daughter cells in only few hours </li></ul><ul><ul><li>remarkably accurate </li></ul></ul><ul><ul><li>only ~1 error per 100 million bases </li></ul></ul><ul><ul><li>~30 errors per cell cycle </li></ul></ul>
  31. 31. What does it really look like? 1 2 3 4
  32. 32. 2007-2008 Any Questions??
  33. 33. Energy of Replication <ul><li>Where does energy for bonding usually come from? </li></ul>energy ATP GTP TTP ATP ADP AMP GMP TMP AMP modified nucleotide We come with our own energy ! And we leave behind a nucleotide ! You remember ATP ! Are there other ways to get energy out of it?
  34. 34. <ul><li>Adding bases </li></ul><ul><ul><li>can only add nucleotides to 3  end of the growing DNA strand </li></ul></ul><ul><ul><ul><li>need a primer nucleotide to bond to </li></ul></ul></ul><ul><ul><li>strand grows 5  3  </li></ul></ul>Replication DNA Polymerase III energy 3  3  5  B.Y.O. ENERGY ! The energy rules the process 5 
  35. 35. 5  3  3  5  3  5  3  5  no energy to bond 
  36. 36. energy 5  3  3  5  3  5  3  5  ligase
  37. 37. Chromosome erosion <ul><li>Loss of bases at 5  ends in every replication </li></ul><ul><ul><li>chromosomes get shorter with each replication </li></ul></ul><ul><ul><li>limit to number of cell divisions? </li></ul></ul>DNA polymerase III DNA polymerases can only add to 3  end of an existing DNA strand growing replication fork DNA polymerase I Houston, we have a problem ! 5  5  5  5  3  3  3  3 
  38. 38. Replication fork 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ direction of replication
  39. 39. DNA synthesis in prokaryotes <ul><li>Replication is bidirectional. </li></ul><ul><li>Replication is semi-conservative. </li></ul>
  40. 40. Bidirectional replication of a circular chromosome Replication begins at the point of origin ( oriC ) and proceeds in both directions at the same time.
  41. 41. Unwinding of Parental Strands Topoisomerases: can break phosphodiester bonds and rejoin them relieve the supercoiling of the parental duplex caused by unwinding . DNA gyrase is a major topoisomerase in bacterial cells.
  42. 42. 以上です。

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