DNA Replication and Nucleotide Structure

1. CHAPTER 16 DNA

2. REVIEW ON ORGANIC MOLECULES • NUCLEOTIDES, THE MONOMERS OF NUCLEIC ACIDS (DNA, RNA) ARE MADE OF 3 SMALLER MOLECULAR BUILDING BLOCKS: • A NITROGENOUS BASE (PURINE OR PYRIMIDINE) • A PENTOSE SUGAR (EITHER DEOXYRIBOSE OR RIBOSE) • A PHOSPHATE GROUP

3. POLYNUCLEOTIDES (DNA) • COVALENT BONDS: CONNECT THE PHOSPHATE GROUPS OF ONE NUCLEOTIDE TO THE SUGAR GROUPS OF NEXT NUCELOTIDE • HYDROGEN BONDS: CONNECT THE NITROGENOUS BASE PAIRS TOGETHER • PURINES (A,G): LARGER, WITH 6-MEMBERED RING FUSED TO 5-MEMBERED RING • PYRIMIDINES (C,T,U): A 6-MEMBERED RING OF CARBON AND NITROGEN ATOMS

4. STRUCTURE OF NUCLEOTIDES

5. WATSON AND CRICK • 1953, DISCOVERED THE DOUBLE HELIX BY BUILDING MODELS TO CONFROM TO X-RAY DATA • THEY FINALLY PROPOSED THE DNA SOLUTION (AFTER MANY UNSUCCESSFUL ATTEMPTS): • DNA HAS A 2 nm WIDTH • A PURINE ON ONE STRAND MUST PAIR WITH A PYRIMIDINE ON THE OTHER • BASE STRUCTURE DICTATES WHICH PAIR OF BASES CAN HYDROGEN BOND: A-T, G-C

6. DNA STRUCTURE

7. VIDEO: DNA/RNA STRUCTURE

8. BASE-PAIRING RULEA-T, G-C • IT EXPLAINS CHARGAFF’S RULES. SINCE A MUST PAIR WITH T, THEIR AMTS. IN A DNA MOLECULE WILL BE THE SAME • IF BASES FORM SPECIFIC PAIRS, THE INFO ON ONE STRAND COMPLEMENTS THAT ALONG THE OTHER • IT DICTATES THE COMBO OF COMPLEMENTARY BASE PAIRS, BUT PLACES NO RESTRICTION ON LINEAR SEQUENCE OF NUCLEOTIDES • THOUGH HYDROGEN BONDS ARE WEAK, THEY COLLECTIVELY STABILIZE THE DNA MOLECULE.

9. THE DOUBLE HELIX

10. BASE PAIRING

11. VIDEO: DNA REPLICATION OVERVIEW

12. DNA REPLICATION • ORIGINS OF REPLICATION: WHERE DNA REPLICATION BEGINS, THERE IS A SPECIFIC SEQUENCE OF NUCELOTIDES • 1) SPECIFIC PROTEINS REQUIRED TO INITIATE REPLICATION BIND TO EACH ORIGIN • 2) THE DOUBLE HELIX OPENS AT THE ORIGIN AND REPLICATION FORKS SPREAD IN BOTH DIRECTIONS AWAY FROM CENTRAL INITIATION POINT CREATING REPLICATION BUBBLE • REPLICATION FORKS - THE Y-SHAPED REGIONS OF REPLICATING DNA MOLEUCLES WHERE NEW STRANDS ARE GRWONING

13. BASIC DNA REPLICATION

14. DNA REPLICATION

15. ELONGATING A NEW DNA STRAND • DNA POLYMERASES - ENZYMES THAT CATALYZE SYNTHESIS OF NEW DNA • ACCORDING TO BASE-PAIR RULES, NEW NUCLEOTIDES ALIGN THEMSELVES ALONG THE TEMPLATES OF THE OLD DNA STRAND • DNA POLMERASE LINKS THE NUCLEOTIDES TO THE GROWING STRAND, IN THE 5’ TO 3’ DIRECTION. • NEW NUCLEOTIDES ARE ONLY ADDED TO THE 3’ END OF THE GROWING STRAND

16. NUCLEOSIDE TRIPHOSPHATE • NUCLEOTIDES WITH A TRIPHOSPAHTE COVALENTLY LINKED TO THE 5’ CARBON OF THE PENTOSE; THESE ARE THE BUIDLING BLOCKS FOR DNA AND THEY LOSE TWO PHOSPHATES WHEN THEY FORM COVALENT LINKAGES TO THE GROWING DNA CHAIN • EXERGONIC HYDROLYSIS OF THIS PHOSPHATE BOND DRIVES THE ENDERGONIC SYNTHESIS OF DNA; IT PROVIDES THE ENERGY NEEDED TO FORM THE NEW LINKAGES BETWEEN NUCLEOTIDES

17. BUILDING A DNA STRAND

18. ANTIPARALLEL DNA STRANDS • BOTH NEW DNA STRANDS CANNOT BE MADE AT THE SAME TIME BECAUSE: • 1) THE SUGAR PHOSPHATE BACKBONES OF THE TWO COMPLEMENTARY DNA STRANDS RUN IN OPPOSITE DIRECTIONS, THAT IS ANTIPARALLEL • 2) RECALL, EACH DNA STRAND HAS A DISTINCT POLARITY. THE 3’ END HAS A HYDROXYL GROUP ATTACHED TO THE 3’ CARBON; THE 5’ END HAS A PHOSPHATE GROUP ATTACHED TO THE 5’ CARBON • 3) DNA POLYMERASE CAN ONLY ELONGATE IN THE 5’ TO 3’ DIRECTION

19. ANTIPARALLEL DNA STRANDS

20. SYNTHESIS OF LEADING STRAND • THE PROBLEM OF ANTIPARALLEL DNA STRANDS IS SOLVED BY THE CONTINUOUS SYNTHESIS OF ONE STRAND (LEADING) AND THE DISCONTINUOUS SYNTHESIS OF THE COMPLEMENTARY STRAND (LAGGING) • LEADING STRAND - MADE IN THE 5’ TO 3’ DIRECTION TOWARDS REPLICATION FORK • LAGGING STRAND - DISCONTINUOUSLY MADE IN OPPOSITE DIRECTION

21. LAGGING STRAND • THE LAGGING STRAND IS MADE AS A SERIES OF SHORT SEGMENTS CALLED OKASAKIFRAGMENTS IN THE 5’ TO 3’ • THE MANY FRAGMENTS ARE LIGATED BY DNA LIGASE, A LINKING ENZYME THAT CATALYZES A COVALENT BOND BETWEEN THE 3’ END OF EACH NEW FRAGMENT TO THE 5’ END OF THE GROWING CHAIN • OKASAKI FRAGMENTS ARE 100 TO 200 NUCLEOTIDES LONG IN EUKARYOTES

22. LEADING AND LAGGING STRANDS

23. SUMMARY OF DNA REPLICATION

24. VIDEO: DNA REPLICATION

25. PRIMING DNA SYNTHESIS • BEFORE NEW DNA STRANDS CAN FORM, THERE MUST BE SMALL PREEXISTING PRIMERS TO START THE ADDITION OF NEW NUCLEOTIDES • PRIMER = SHORT RNA SEGMENT THAT IS COMPLEMENTARY TO A DNA SEGMENT AND THAT IS NEEDED TO BEGIN REPLICATION

26. PRIMERS • SHORT SEGMENTS OF RNA POLYMERIZED BY AN ENZYME CALLED PRIMASE • A PORTION OF PARENTAL DNA SERVES AS A TEMPLATE FOR MAKING THE PRIMER WITH A COMPLEMENTARY BASE SEQUENCE OF ABOUT TEN NUCLEOTIDES • PRIMER FORMATION MUST PRECEDE DNA REPLICATION, BECAUSE DNA POLYMERASE CAN ONLY ADD NUCLEOTIDES TO A STRAND THAT IS CORRECTLY BASE-PAIRED WITH A COMPLEMENTARY STRAND

27. PRIMING DNA SYNTHESIS

28. PRIMERS FOR LAGGING STRAND • MANY PRIMERS ARE NEEDED TO REPLICATE LAGGING STRAND BECAUSE A DIFFERENT RNA PRIMER MUST INITIATE EACH OKAZAKI FRAGMENT

29. OTHER PROTEINS INVOLVED • HELICASES - ENZYMES WHICH CATALYZE UNWINDING OF THE PARENTAL DOUBLE HELIX • SINGLE-STRAND BINDING PROTEINS - KEEP THE SEPARATED STRANDS APART AND STABILIZED UNWOUND DNA

30. THE PROTEINS OF DNA REPLICATION

31. PROOFREADING DNA REPLICATION • MISMATCH REPAIR - CORRECTS MISTAKES IN INCORRECTLY PAIRED NUCLEOTIDES; DNA POYMERASE PROOFREADS NEW STRANDS AND CORRECTS PROBLEMS .EXCISION REPAIR - THERE ARE MORE THAN 50 DIFFERENT TYPE OF DNA REPAIR ENZYMES THAT CAN EXISE PROBLEM AND FILL IN REMAINING GAP BY BASE-PAIRING NUCLEOTIDES WITH THE UNDAMAGED STRAND

32. EXCISION REPAIR OF DNA DAMAGE

33. THE END-REPLICATION PROBLEM • THE FACT THAT A DNA POLYMERASE CAN ONLY ADD NUCLEOTIDES TO THE 3’ END OF A PREEXISITING STRAND CREATES A PROBLEM • THE USUAL REPLICATION MACHINERY PROVIDES NO WAY TO COMPLETE THE 5’ ENDS OF THE DAUGHTER DNA STRANDS, RESULTING IN SHORTER AND SHORTER DNA MOLECULES

34. THE END REPLICATION PROBLEM

35. THE SOLUTION: TELOMERASES • TELOMERASES - REPEATS OF SHORT NONCODING NUCLEOTIDES SEQUENCES • THESE ENZYMES HAVE A SHORT MOLECULE OF RNA WITH A SEQUENCE THAT SERVES AS A TEMPLATE FOR EXTENDING THE 3’ END OF THE TELOMERE

36. TELOMERASE