2. AP Biology 2007-2008 DNA Replication

3. STRUCTURE OF NUCLEIC ACIDS Sugar can be DEOXYRIBOSE (DNA) RIBOSE (RNA) Built from NUCLEOTIDE SUBUNITS NITROGEN BASES CAN BE: ADENINE GUANINE CYTOSINE THYMINE URACIL

4. DNA has no URACILRNA has no THYMINE PURINES (A & G) have 2 RINGS PYRIMIDINES (T, C, & U) have 1 RING

5. 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

6. AP Biology The DNA backbone  Made of phosphates and deoxyribose sugars  Phosphate on 5’ carbon attaches to 3’ carbon of next nucleotide 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

7. 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

8. 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

9. 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

10. 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

11. AP Biology CHARGAFF’s RULES Erwin Chargaff analyzed DNA from different organisms and found A = T G = C Now know its because: A always bonds with T G always bonds with C A Purine always bonds to a Pyrimidine

12. AP Biology Semi- Conservative Dispersive

13. AP Biology Chromosome E. coli bacterium Bases on the chromosome Chromosome Structure in Prokaryotes © Pearson Education Inc, publishing as Pearson Prentice Hall. All rights reserved DNA molecule in bacteria single DOUBLE STRANDED circular loop Approximately 5 million base pairs 3,000 genes

14. AP Biology Starting place = ORIGIN OF REPLICATION Bacteria have one Bacterial replication Eukaryotes- multiple origins

15. AP Biology HOW NUCLEOTIDES ARE ADDED DNA REPLICATION FORK DNA replication Triphosphate addition DNA replication 2 DNA replication/quiz

16. AP Biology 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  semi-conservative copy process

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 DNA REPLICATION FORK

18. AP Biology DNA Polymerase III Replication: 2nd step Where ’ s the ENERGY for the bonding come from?  Build daughter DNA strand  add new complementary bases  DNA polymerase III

19. AP Biology energy ATP Energy of Replication Where does energy for bonding usually come from? ADP modified nucleotide We come with our own energy!

20. AP Biology ATP Energy of Replication Where does energy for bonding usually come from? AMP modified nucleotide energy We come with our own energy! And we leave behind a nucleotide! Are there other energy nucleotides? You bet! DNA replication

21. AP Biology Energy of Replication  The nucleotides arrive as nucleoside triphosphates  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 See animation

22. 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 Replication 3 3 5 5 need “primer” bases to add on to

23. AP Biology DNA Polymerase III energy Replication 3 3 5 5

24. AP Biology DNA Polymerase III energy Replication 3 3 5 5

25. AP Biology Replication 3 3 5 5

26. AP Biology 35 5 5 3 need “primer” bases to add on to 3 energy 35 Can’t build 3’ to 5’ direction

27. AP Biology 35 5 5 3 3 35 need “primer” bases to add on to

28. AP Biology 35 5 5 3 need “primer” bases to add on to 3 energy 35

29. AP Biology 35 5 5 3 need “primer” bases to add on to 3 energy 35

30. AP Biology 35 5 5 3 3 35

31. 35 5 5 3 3 energy 35

32. AP Biology 35 5 5 3 3 35 ligase Joins fragments

33. 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

34. AP Biology DNA polymerase III Replication fork 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

35. 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

36. 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

37. 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

38. TELOMERES & TELOMERASE Image from: AP BIOLOGY by Campbell and Reese 7 th edition Primer removed but can’t be replaced with DNA because no 3’ end available for DNA POLYMERASE Each replication shortens DNA strand

39.  TELOMERES-repetitive sequences added to ends of genes to protect information in code TELOMERES  TELOMERASE can add to telomere segments in cells that must divide frequently  Shortening of telomeres may play a role in aging  Cells with increased telomerase activity which allows them to keep dividing EX: Cells that give rise to sperm & eggs, stem cells, cancer cells ANIMATION

40. 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

41. 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 Thomas Kornberg

42. 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

43. 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

44. PROOFREADING & REPAIR Errors can come from:  “proofreading mistakes” that are not caught  Environmental damage from CARCINOGENS (Ex: X-rays, UV light, cigarette smoke, etc) EX: Thymine dimersThymine dimers

45. NUCLEOTIDE EXCISION REPAIR  Cells continually monitor DNA and make repairs  NUCLEASES-DNA cutting enzyme removes errors  DNA POLYMERASE AND LIGASE can fill in gap and repair using other strand  Xeroderma pigmentosum- genetic disorder  mutation in DNA enzymes that repair UV damage in skin cells  can’t go out in sunlight  increased skin cancers/cataracts