2. AP Biology 2007-2008 Synthesis of DNA June.21.2010

3. AP Biology 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.

4. AP Biology Each molecule generated by the replication process contains one intact parental strand and one newly synthesized strand.

5. AP Biology 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.

6. AP Biology Cultured MDCK cells Day 1 Day 3

7. AP Biology Watson and Crick 1953 article in Nature

8. AP Biology 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

9. AP Biology Directionality of DNA  You need to number the carbons!  it matters! OH CH 2 O 4 5 3 2 1 PO 4 N base ribose nucleotide This will be IMPORTANT!!

10. AP Biology The DNA backbone  Putting the DNA backbone together  refer to the 3 and 5 ends of the DNA  the last trailing carbon Sounds trivial, but … this will be IMPORTANT!! OH O 3 PO 4 base CH 2 O base O P O C O –O–O CH 2 1 2 4 5 1 2 3 3 4 5 5

11. AP Biology 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 3 5 5 3

12. AP Biology 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

13. AP Biology Base pairing in DNA  Purines  adenine (A)  guanine (G)  Pyrimidines  thymine (T)  cytosine (C)  Pairing  A : T  2 bonds  C : G  3 bonds

14. AP Biology

15. 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

16. AP Biology DNA Replication  Large team of enzymes coordinates replication Let ’ s meet the team …

17. AP Biology Replication: 1st step  Unwind DNA  helicase enzyme  unwinds part of DNA helix  stabilized by single-stranded binding proteins single-stranded binding proteins replication fork helicase

18. AP Biology Definitions  Template strands: The old strands which are now separated from each other.  New strands: The growing complementary nucleotide sequences that allow for forming the two double helix structures  Note that replication is direction specific.

19. AP Biology DNA Elongation  Works only 5’ to 3’  This means that the 5’ end of the nucleotide links on to the 3’ end of the DNA (or RNA primer)  The NEW strand only grows 5’ to 3’ OH O 3 PO 4 base CH 2 O base O P O C O –O–O CH 2 1 2 4 5 1 2 3 3 4 5 5

20. AP Biology DNA polymerase III RNA primer  built by primase  serves as starter sequence for DNA polymerase III Limits of DNA polymerase III  can only build onto 3 end of an existing DNA strand Starting DNA synthesis: RNA primers 5 5 5 3 3 3 5 3 5 3 5 3 growing replication fork primase RNA

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

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

23. AP Biology DNA polymerase III Replication fork / Replication bubble 5 3 5 3 leading strand lagging strand leading strand lagging strand leading strand 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 lagging strand 5 3

24. AP Biology DNA Polymerase III Replication: 2nd step But … We ’ re missing something! What? Where ’ s the ENERGY for the bonding!  Build daughter DNA strand  add new complementary bases  DNA polymerase III

25. AP Biology DNA polymerase I  removes sections of RNA primer and replaces with DNA nucleotides But DNA polymerase I still can only build onto 3 end of an existing DNA strand Replacing RNA primers with DNA 5 5 5 5 3 3 3 3 growing replication fork DNA polymerase I RNA ligase

26. AP Biology Fast & accurate!  It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome  divide to form 2 identical daughter cells  Human cell copies its 6 billion bases & divide into daughter cells in only few hours  remarkably accurate  only ~1 error per 100 million bases  ~30 errors per cell cycle

27. AP Biology 1 2 3 4 What does it really look like?

28. AP Biology

29. energy ATP GTPTTPCTP Energy of Replication Where does energy for bonding usually come from? ADPAMPGMPTMPCMP 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!

30. AP Biology 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  bonded by enzyme: DNA polymerase III ATPGTPTTPCTP

31. AP Biology  Adding bases  can only add nucleotides to 3 end of a growing DNA strand  need a “starter” nucleotide to bond to  strand only grows 5  3 DNA Polymerase III DNA Polymerase III DNA Polymerase III DNA Polymerase III energy Replication energy 3 3 5 B.Y.O. ENERGY! The energy rules the process 5

32. AP Biology energy 35 5 5 3 need “primer” bases to add on to energy 3 no energy to bond energy ligase 35 

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

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

35. AP Biology 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 Arthur Kornberg 1959 Roger Kornberg 2006

36. AP Biology Editing & proofreading DNA  1000 bases/second = lots of typos!  DNA polymerase I  proofreads & corrects typos  repairs mismatched bases  removes abnormal bases  repairs damage throughout life  reduces error rate from 1 in 10,000 to 1 in 100 million bases

37. AP Biology 2007-2008 Any Questions??

38. AP Biology energy ATP GTPTTPATP Energy of Replication Where does energy for bonding usually come from? ADPAMPGMPTMPAMP 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?

39. AP Biology  Adding bases  can only add nucleotides to 3 end of the growing DNA strand  need a primer nucleotide to bond to  strand grows 5  3 DNA Polymerase III Replication energy 3 3 5 B.Y.O. ENERGY! The energy rules the process 5

40. AP Biology 5 3 3 5 35 35 no energy to bond 

41. AP Biology energy 5 3 3 5 35 35 ligase

42. AP Biology Loss of bases at 5 ends in every replication  chromosomes get shorter with each replication  limit to number of cell divisions? DNA polymerase III DNA polymerases can only add to 3 end of an existing DNA strand Chromosome erosion 5 5 5 5 3 3 3 3 growing replication fork DNA polymerase I Houston, we have a problem!

43. AP Biology Replication fork 3’ 5’ 3’ 5’ 3’ 5’ direction of replication

44. AP Biology DNA synthesis in prokaryotes 1.Replication is bidirectional. 2.Replication is semi-conservative.

45. AP Biology Bidirectional replication of a circular chromosome Replication begins at the point of origin (oriC) and proceeds in both directions at the same time.

46. AP Biology 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.

47. AP Biology 以上です。