Lecture 1:

1. Lecture 1: DNA Polymerase and the Replication Fork Use of biochemistry (assays) and genetics (mutant phenotypes) to define function Fidelity/Specificity: bioregulation through substrate control of molecular choice

2. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

3. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

4. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

5. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

6. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

7. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

8. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

9. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

10. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene mutation by HR)

11. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene mutation by HR) expression interference (siRNA, shRNA)

12. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene mutation by HR, CRISPR/Cas9) expression interference (siRNA, shRNA, CRISPRi)

13. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

14. DNA Replication: The Task and Challenge Semiconservative Duplication Speed: very rapid duplication of every nucleotide (ex: 6 x 109 bp in 8 hrs in humans) Fidelity: extremely low error rate (~1/109 nucleotide error rate) Count: exactly two copies of every sequence per cell cycle Regulation: coordination with other chromosomal events (eg.mitosis, repair, recombination, transcription, chromatin packaging)

15. Enzymology of DNA Synthesis: DNA Polymerases dNTP precursor - pyrophosphate release provides energy Instructed by single-stranded template - senses complementarity of new nucleotide Primer requirement* - senses complementarity of primer 5’ > 3’ polymerization off primer* - extension off 3’ hydroxyl - moving 3’> 5’ on template * enhances fidelity by allowing error correction

16. Initial conditions used were really assaying a complex mixture of activities: E. coli extract - source of polymerase activity but also kinase and nuclease activity 14C Thymidine - converted to thymidine triphosphate by kinases in extract DNA - intended as nuclease decoy but nucleases convert to primer-template and source of A,G,C nucleotides Assaying DNA Polymerase Activity In principle: Monitor incorporation of radioactive nucleotide precursors ( ) into acid insoluble form (physically separate product from precursor) First Experiment: 50 out of 1 million cpm insoluble Ten Years Later: purify DNA Polymerase I, show it is template-directed and figure out enzyme requirements

17. Initial conditions used were really assaying a complex mixture of activities: E. coli extract - source of polymerase activity but also kinase and nuclease activity 14C Thymidine - converted to thymidine triphosphate by kinases in extract DNA - intended as nuclease decoy but nucleases convert to primer-template and source of A,G,C nucleotides Assaying DNA Polymerase Activity In principle: Monitor incorporation of radioactive nucleotide precursors ( ) into acid insoluble form (physically separate product from precursor) In practice: Can be difficult to devise the right assay conditions when you do not know the precise nature of the activity First Experiment: 50 out of 1 million cpm insoluble Ten Years Later: purify DNA Polymerase I, show it is template-directed and figure out enzyme requirements

18. Assaying DNA Polymerase Activity In principle: Monitor incorporation of radioactive nucleotide precursors ( ) into acid insoluble form (physically separate product from precursor) In practice: Can be difficult to devise the right assay conditions when you do not know the precise nature of the activity E. coli extract: source of polymerase activity 14C Thymidine: nucleotide precursor DNA: intended as nuclease decoy

19. Assaying DNA Polymerase Activity In principle: Monitor incorporation of radioactive nucleotide precursors ( ) into acid insoluble form (physically separate product from precursor) In practice: Can be difficult to devise the right assay conditions when you do not know the precise nature of the activity E. coli extract: source of polymerase activity but also kinase and nuclease activity 14C Thymidine: nucleotide precursor converted to true precursor, thymidine triphosphate, by kinases in extract DNA: intended as nuclease decoy, but nucleases convert to primer- template and source of A,G,C nucleotides

20. Initial conditions used were really assaying a complex mixture of activities: E. coli extract: source of polymerase activity but also kinase and nuclease activity 14C Thymidine: nucleotide precursor converted to true precursor, thymidine triphosphate, by kinases in extract DNA: intended as nuclease decoy, but nucleases convert to primer- template and source of A,G,C nucleotides Assaying DNA Polymerase Activity In principle: Monitor incorporation of radioactive nucleotide precursors ( ) into acid insoluble form (physically separate product from precursor) In practice: Can be difficult to devise the right assay conditions when you do not know the precise nature of the activity First Experiment: 50 out of 1 million cpm insoluble

21. Initial conditions used were really assaying a complex mixture of activities: E. coli extract: source of polymerase activity but also kinase and nuclease activity 14C Thymidine: nucleotide precursor converted to true precursor, thymidine triphosphate, by kinases in extract DNA: intended as nuclease decoy, but nucleases convert to primer- template and source of A,G,C nucleotides Assaying DNA Polymerase Activity In principle: Monitor incorporation of radioactive nucleotide precursors ( ) into acid insoluble form (physically separate product from precursor) In practice: Can be difficult to devise the right assay conditions when you do not know the precise nature of the activity First Experiment: 50 out of 1 million cpm insoluble Ten Years Later: purify DNA Polymerase I, show it is template-directed and figure out enzyme requirements

22. DNA Polymerase Structure and Catalysis Crystal structure of bacteriophage T7 DNA Polymerase complexed with primer-template and dNTP Two Mg++ ions positioned by conserved acidic residues catalyze reaction Incoming Nucleotide Primer Template Rest of enzyme positions primer-template and dNTP and ensures catalysis only occurs with proper “fit” Structure resembles a right hand

23. DNA Pol I has 3’ > 5’ Exonuclease Activity Careful quantitative analysis of biochemical activity can suggest biological function * T * T T T T T T T 3’ 5’ Exo Assay: DNA Pol I 5’ 3’ AAAAAAAA AAAAAAAA 5’ 5’ 3’ 3’ no dTTP *

24. exo activity is slow relative to pol activity exo activity is enhanced by stalling pol activity or making 3’ end single-stranded 3’ mismatch generates both conditions DNA Pol I has 3’ > 5’ Exonuclease Activity Careful quantitative analysis of biochemical activity can suggest biological function * T * T T T T T T T 3’ 5’ Exo Assay: DNA Pol I 5’ 3’ AAAAAAAA AAAAAAAA 5’ 5’ 3’ 3’ no dTTP *

25. exo activity is slow relative to pol activity exo activity is enhanced by stalling pol activity or making 3’ end single-stranded 3’ mismatch generates both conditions * T T T T T T T T T T T T Proofread Assay: 3’ 5’ 5’ DNA Pol I 3’ AAAAAAAA AAAAAAAA 5’ 3’ 5’ 3’ + dTTP * T T T T T T T T T T C T 3’ 5’ 5’ DNA Pol I 3’ AAAAAAAA AAAAAAAA 5’ 3’ 5’ 3’ + dTTP * C mismatch specific exo activity under normal pol conditions both pol and exo activities are sensing primer-template pairing DNA Pol I has 3’ > 5’ Exonuclease Activity Careful quantitative analysis of biochemical activity can suggest biological function * T * T T T T T T T 3’ 5’ Exo Assay: DNA Pol I 5’ 3’ AAAAAAAA AAAAAAAA 5’ 5’ 3’ 3’ no dTTP *

26. The Polymerase and Exonuclease Activities of Replicative DNA Polymerases Reside in Distinct Domains Polymerase Active Site ~ 40Å Exonuclease Active Site

27. 2- Mode Model for Polymerase Function Polymerizing Editing Movement between P and E sites requires primer-template unwinding translocation of 3’ end The Polymerase and Exonuclease Activities of Replicative DNA Polymerases Reside in Distinct Domains Polymerase Active Site ~ 40Å Exonuclease Active Site

28. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

29. DNA Pol I is not the replicative DNA polymerase in E. coli Illustrates importance of genetics for establishing functional relevance in cell Use biochemical assay to screen for mutants lacking DNA polymerase activity plate mutagenize phenotype: assay dNTP incorporation into DNA mutant E. coli E. coli extracts from single mutant colonies

30. DNA Pol I is not the replicative DNA polymerase in E. coli Illustrates importance of genetics for establishing functional relevance in cell Use biochemical assay to screen for mutants lacking DNA polymerase activity plate mutagenize phenotype: assay dNTP incorporation into DNA mutant E. coli E. coli extracts from single mutant colonies mutant 3473 (polA1) has <1% wt activity normal growth repair deficient

31. DNA Pol I is not the replicative DNA polymerase in E. coli Illustrates importance of genetics for establishing functional relevance in cell Use biochemical assay to screen for mutants lacking DNA polymerase activity plate mutagenize phenotype: assay dNTP incorporation into DNA mutant E. coli E. coli extracts from single mutant colonies mutant 3473 (polA1) has <1% wt activity normal growth repair deficient Purification of residual polymerase activity from polA1 yields DNA Pol II and Pol III Genetics and biochemistry later show: - DNA Pol III is the replicative polymerase - DNA Pol I is important for okazaki fragment maturation

32. Purification of DNA Pol III: Different Template, Different Assay, Different Activity Introducing the concept of holoenzymes and modular enzyme subassemblies

33. Purification of DNA Pol III: Different Template, Different Assay, Different Activity Introducing the concept of holoenzymes and modular enzyme subassemblies

34. Using both Biochemistry and Genetics to understand function reverse genetics (targeted gene disruption by HR, ZFN, TALEN, CRISPR/Cas9) expression interference (RNAi, siRNA, CRISPRi)

35. Bioregulation Controlling Molecular Choice

36. Fidelity Overview Fidelity Comparisons Error Rate Product Size Speed DNA Replication 500 bp/sec 5 x 106 10-9 - 10-10 (Prokaryotes) (E. coli) 6 x 109 50 bp/sec (humans) (Eukaryotes) 1 x 1011 (lily) RNA Transcription 30 bp/sec 10-4 103 - 106 Protein Translation 20 aa/sec 10-4 102 - 103

37. Fidelity Overview Contributions to E coli DNA Replication Fidelity Fidelity Comparisons Error Rate Product Size Speed Error rate DNA Replication Intrinsic Fidelity (polym) 10-3 - 10-4 500 bp/sec 5 x 106 10-9 - 10-10 (Prokaryotes) (E. coli) (sensing dNTP complementarity to template) 6 x 109 50 bp/sec (humans) (Eukaryotes) Exonuclease Proofreading (polym) 10-2 - 10-3 (sensing primer complementarity to template) 1 x 1011 (lily) Mismatch Repair (post polym) 10-2 RNA Transcription 30 bp/sec 10-4 103 - 106 (sensing complementarity of two strands) (distinguishing parental and daughter strands) Protein Translation 20 aa/sec 10-4 102 - 103 Overall Replication Fidelity 10-8 - 10-9

38. Sources of Polymerase Discrimination H-bonding (binding energetics) Outside the active site, unpaired nucleotides are H-bonded to H2O. Inside the active site these H-bonds can be replaced by WC base pairing but only incompletely replaced by mismatch pairing Mismatch H bonding can also exacerbate steric and stacking clashes (see below) Steric Constraints (structure/geometry) Imposed by enzyme’s “induced fit”, which can test for precise Watson-Crick base pair geometry and proper base pair stacking How to Distinguish Mismatch versus Correct Base Pair

39. Sources of Polymerase Discrimination H-bonding (binding energetics) Outside the active site, unpaired nucleotides are H-bonded to H2O. Inside the active site these H-bonds can be replaced by WC base pairing but only incompletely replaced by mismatch pairing Mismatch H bonding can also exacerbate steric and stacking clashes (see below) Steric Constraints (structure/geometry) Imposed by enzyme’s “induced fit”, which can test for precise Watson-Crick base pair geometry and proper base pair stacking How to Distinguish Mismatch versus Correct Base Pair

40. Sources of Polymerase Discrimination H-bonding (binding energetics) Outside the active site, unpaired nucleotides are H-bonded to H2O. Inside the active site these H-bonds can be replaced by WC base pairing but only incompletely replaced by mismatch pairing Mismatch H bonding can also exacerbate steric and stacking clashes (see below) Steric Constraints (structure/geometry) Imposed by enzyme’s “induced fit”, which can test for precise Watson-Crick base pair geometry and proper base pair stacking How to Distinguish Mismatch versus Correct Base Pair Base Pair Geometry: WC vs Mismatch WC bp mismatch WC bp mismatch mismatch Global structure of helix is not greatly perturbed But there are: differences in C1’ - C1’ distance and C1’ bond angles protrusions of bases into major groove loss of universal H acceptor positions in minor groove

41. C C k k pol conf C K C dNTP E DNA D N C E DNA PP N+1 i * C dNTP E DNA N 1 3 2 E DNA N I I k k pol conf I K D I PP E DNA N+1 i * I dNTP E DNA N I dNTP E DNA N Intrinsic Fidelity: Potential Base Pair Discrimination for dNTP at Three Stages Of the DNA Polymerization Reaction Cycle Example: T7 DNA polymerase (arrow thickness roughly corresponds to rate CONSTANTS) Other polymerases discriminate differently at each stage Reaction pathway for correct nucleotide Reaction pathway for incorrect nucleotide Rapid dNTP Binding Pseudo-equilibrium Slow Conformational Change “Induced Fit” Polymerization Reaction

42. C C C k k k pol conf conf C C K K C dNTP D E DNA D N C E DNA PP N+1 i * C dNTP E DNA N 1 3 2 E DNA N I I I k k k pol conf conf I K D I K I PP E DNA D N+1 i * I dNTP E DNA N I dNTP E DNA N Intrinsic Fidelity: Potential Base Pair Discrimination for dNTP at Three Stages Of the DNA Polymerization Reaction Cycle Example: T7 DNA polymerase (arrow thickness roughly corresponds to rate CONSTANTS) Other polymerases discriminate differently at each stage Reaction pathway for correct nucleotide Reaction pathway for incorrect nucleotide Rapid dNTP Binding Pseudo-equilibrium Slow Conformational Change “Induced Fit” Polymerization Reaction -1 > 8mM 300 s ~ ~ ~ ~ 1000x 400x Rapid and Not Measured -1 20 µM 0.3 s

43. C E DNA PP N+1 i I PP E DNA N+1 i

44. C E DNA N+1 I E DNA N+1

45. C C k k pol conf C E DNA PP C C K K N+2 i C dNTP E DNA D D N Fast reaction pathways for correct primer with correct nucleotide * C dNTP E DNA N 1 3 2 Slow reaction pathways for incorrect primer with correct nucleotide I C I C I k k conf pol Rapid dNTP Binding Pseudo-equilibrium Slow Conformational Change “Induced Fit” Polymerization Reaction C E DNA N+1 I * C C E dNTP dNTP DNA I I E DNA E DNA N+1 N+1 N+1 I E DNA PP N+2 i

46. C C k k pol conf C E DNA PP C C K K N+2 i C dNTP E DNA D D N * C dNTP E DNA N 1 3 2 I C I C I k k conf pol C E DNA N+1 I * C C E dNTP dNTP DNA I I E DNA E DNA N+1 N+1 N+1 Error Correction: Primer requirement allows kinetic discrimination sensitive to base pairing of recently incorporated nucleotides Fast reaction pathways for correct primer with correct nucleotide Slow reaction pathways for incorrect primer with correct nucleotide I E DNA PP N+2 i Rapid dNTP Binding Pseudo-equilibrium Slow Conformational Change “Induced Fit” Polymerization Reaction

47. C C E E DNA DNA C C C C k k k k k N N exo pol pol conf conf C C E E DNA DNA PP PP C C C K K K N+2 N+2 i i C C dNTP dNTP E E DNA DNA D D D N N * * C C dNTP dNTP E E DNA DNA N N 1 3 2 I C I C I k k conf pol I k exo C C E E DNA DNA N+1 N+1 I E DNA PP I * C C N+2 i E dNTP dNTP DNA I I E DNA E DNA N+1 N+1 N+1 Error Correction: Exonuclease activity allows the polymerase’s kinetic discrimination to lead to different primer fates Arrow thickness roughly corresponds to rate constant Fast reaction pathways for correct primer with correct nucleotide Slow reaction pathways for incorrect primer with correct nucleotide

48. C C E E DNA DNA N N C E DNA PP N+2 i C E DNA N+1 I E DNA PP N+2 i I E DNA N+1 Error Correction: Kinetic manipulation of molecular choice based on complementarity of primer Arrow thickness roughly corresponds to rate CONSTANT When a correct nucleotide is incorporated, 3’>5’ exonuclease activity is much slower than 5’>3’ polymerase activity. Addition of the next nucleotide is kinetically favored. exo pol pol When an incorrect nucleotide is incorporated, disruption of the primer greatly slows 5’>3’ polymerase activity for the next nucleotide (and slightly increases 3’>5’ exonuclease activity). Excision of the incorrect nucleotide is kinetically favored. exo

49. 5’-TAGCTTC 5’-TAGCTTC 5’-TAGCTTCA 5’-TAGCTTC 5’-TAGCTTCGA 5’-TAGCTTCG Example: T7 DNA Polymerase 3’-ATCGAAGCTCATG 3’-ATCGAAGCTCATG 3’-ATCGAAGCTCATG 3’-ATCGAAGCTCATG 3’-ATCGAAGCTCATG 3’-ATCGAAGCTCATG A ~ 1500 A ~ 1 230 C primer C primer I primer I primer k k k k T7pol T7pol T7pol T7pol C primer C primer I primer I primer k k k k T7exo T7exo T7exo T7exo -1 -1 -1 -1 0.01 s 300 s 0.2 s 2.3 s Error Correction: Kinetic manipulation of molecular choice based on complementarity of primer Black arrow thickness roughly corresponds to relative rate constant Light blue arrow thickness roughly corresponds to relative flux exo pol pol exo

50. A Discard Strategy for Fidelity: Kinetic manipulation of molecular choice between irreversible forward and discard pathways The choice is ultimately determined by the relative flux of molecules that proceed down the two competing pathways (light blue arrow) Elimination Pathway irreversibility usually requires some chemical energy expenditure (e.g dNTP hydrolysis), which could be coupled to either pathway or to a reaction step preceding these pathways discard forward Cognate Substrate Correct Product forward Noncognate Substrate Incorrect Product For each substrate, the molecular flux (and hence molecular choice) is determined by the ratio of the forward to discard rate constants (black arrows) for that substrate. For cognate substrates this ratio should favor the forward reaction. For noncognate substrates, the ratio should “flip” to favor the discard pathway. discard Elimination In principal, just one or both pathways could discriminate between cognate and noncognate substrates, i.e. change rate constants with substrate. In practice, nature often discriminates with both.