Exploring DNA Structure & Function: Essential Concepts in Biochemistry

1. Biochemistry of Medicinals I – Nucleic Acids Instructor: Natalia Tretyakova, Ph.D. 760E CCRB (Cancer Center) Tel. 6-3432 e-mail trety001@umn.edu Lecture: MWF 3:35-4:25 7-135 WDH Recitation: Th. 11-12 Web page: see “Web enhanced courses”

3. Chapter 1. DNA Structure. Required reading: Stryer 5th Edition p. 117-125, 144-146, 152, 746-750, 754-762, 875-877) (or Stryer’s Biochemistry 4th edition p. 75-77,80-88, 119-122, 126-128, 787-799, 975-980)

4. DNA Structure: Chapter outline • Biological roles of DNA. Flow of genetic information. • Primary and secondary structure of DNA. • Types of DNA double helix. Sequence-specific DNA recognition by proteins. • Biophysical properties of DNA. • DNA topology. Topoisomerases. • Restriction Endonucleases. Molecular Cloning

5. (ribonucleic acids) (deoxyribonucleic acids) replication transcription translation DNA

6. Why ? • Questions? • How is genetic information transmitted to progeny cells? • How is DNA synthesis initiated? • What causes DNA defects and what are their biological an physiological consequences? • What causes the differences between cells containing the same genetic information? • Relevance: • •Cancer: ex., Xeroderma pigmentosum • •Genetic diseases: ex., cystic fibrosis, sickle cell anemia, inborn errors of metabolism • •Genetic typing: ex., drug metabolism • •Rational drug design: ex., antitumor and antimicrobial drugs • •Biotechnology: ex., growth hormones

7. The Building Blocks of DNA -N-glycosidic bond

8. DNA and RNA nucleobases (DNA only) (RNA only)

9. Purine Nucleotides

10. Pyrimidine Nucleotides

11. Example: nucleobase Adenine

12. Nucleoside 2’-deoxyadenosine

13. Nucleotide 2’-deoxyadenosine-5’-monophosphate

14. nucleobase (Deoxy) nucleoside 5’-mononucleotide Adenine (A) Guanine (G) Thymine (T) Cytosine (C) Uracil (U) 2’-Deoxyadenosine (dA) 2’- Deoxyguanosine (dG) 2’- Deoxythymidine (dT) 2’- Deoxycytidine (dC) Uridine (U) Deoxyadenosine 5’-monophosphate (5’-dAMP) Deoxyguanosine 5’-monophosphate (5’-dGMP) Deoxythymidine 5’-monophosphate (5’-dTMP) Deoxycytidine 5’-monophosphate (5’-dCMP) Uridine 5’-monophosphate (5’-UMP) Nomenclature of nucleobases, nucleosides, and mononucleotides

15. Structural differences between DNA and RNA DNA RNA

16. Preferred conformations of nucleobases and sugars in DNA and RNA Sugar puckers: 5.9 A 7.0 A

17. Nucleosides Must Be Converted to5’-Triphosphates to be Part of DNA and RNA

18. DNA isArranged5’ to 3’Connected byPhosphates Linking inDNA biopolymer: DNA primary structure

19. DNA secondary structure – double helix • James Watson and Francis Crick, 1953- proposed a model for DNA structure • DNA is the molecule of heredity (O.Avery, 1944) • X-ray diffraction (R.Franklin and M. Wilkins) • E. Chargaff (1940s) G = C and A = T in DNA Francis Crick Jim Watson

20. Watson-Crick model of DNA was based on X-ray diffraction picture of DNA fibres (Rosalind Franklin and Maurice Wilkins) Rosalind Franklin

21. Watson-Crick model of DNA was consistent with Chargaff’s base composition rules Erwin Chargaff (Columbia University) G = C and A = T in DNA

22. Living Figure – B-DNA http://bcs.whfreeman.com/biochem5

23. DNA is Composed of Complementary Strands

24. Base Pairing is Determined by Hydrogen Bonding same size

25. Base stacking: an axial view of B-DNA

26. Forces stabilizing DNA double helix • Hydrogen bonding (2-3 kcal/mol per base pair) • Stacking (hydrophobic) interactions • (4-15 kcal/mol per base pair) • 3. Electrostatic forces.

27. B-DNA • •Sugars are in the 2’ endo conformation. • •Bases are the anti conformation. • •Bases have a helical twist of 36º • (10 bases per helix turn) • Helical pitch = 34 A 23.7 A right handed helix • helical axis passes through • base pairs 7.0 A • planes of bases are nearly • perpendicular to the helix axis. • 3.4 A rise between base pairs Wide and deep Narrow and deep

28. DNA can deviate from Ideal Watson-Crick structure • Helical twist ranges from 28 to 42° • Propeller twisting 10 to 20° • Base pair roll

29. N NH 2 H N O 2 N N HN C-1’ N N NH O 2 C-1’ Major and minor groove of the double helix O N NH N N N N C-1’ O C-1’ Wide and deep Narrow and deep

30. Major groove and Minor groove of DNA N NH O 2 N H N O 2 N NH N N N HN C-1’ N N N N C-1’ NH O O 2 C-1’ Hypothetical situation: the two grooves would have similar size if dR residues were attached at 180° to each other To deoxyribose-C1’ C1’ -To deoxyribose C-1’

31. B-type duplex is not possible for RNA steric “clash”

32. A-form helix:dehydrated DNA; RNA-DNA hybrids • •Sugars are in the 3’ endo conformation. • •Bases are the anti conformation. • •11 bases per helix turn • Helical pitch = 25.3 A Right handed helix • planes of bases are tilted • 20 ° relative the helix axis. • 2.3 A rise between base pairs 25.5 A Top View

33. Living Figure – A-DNA http://bcs.whfreeman.com/biochem5

34. The sugar puckering in A-DNA is 3’-endo 5.9 A 7.0 A

35. A-form helix:dehydrated DNA; RNA-DNA hybrids • •Sugars are in the 3’ endo conformation. • •Bases are the anti conformation. • •11 bases per helix turn • Helical pitch = 25.3 A Right handed helix • planes of bases are tilted • 20 ° relative the helix axis. • 2.3 A rise between base pairs 25.5 A Top View

36. A-DNA has a shallow minor groove and a deep major groove • • Helix axis A-DNA B-DNA

37. Z-form double helix:polynucleotides of alternating purines and pyrimidines (GCGCGCGC) at high salt • • Backbone zig-zags because sugar puckers alternate between 2’ endo pyrimidines and 3’ endo (purines) • • Bases alternate between anti (pyrimidines) and syn conformation (purines). • •12 bases per helix turn • Helical pitch = 45.6 A Left handed helix • planes of the bases are • tilted 9° relative the helix • axis. • 3.8 A rise between base pairs 18.4 A • Flat major groove • Narrow and deep minor groove

38. Sugar and base conformations in Z-DNA alternate: 5’-GCGCGCGCGCGCG 3’-CGCGCGCGCGCGC C:sugar is 2’-endo, base is anti G: sugar is 3’-endo, base is syn

39. Living Figure – Z-DNA http://bcs.whfreeman.com/biochem5

41. Biological relevance of the minor types of DNA secondary structure • Although the majority of chromosomal DNA is in B-form, • some regions assume A- or Z-like structure • Runs of multiple Gs are A-like • The upstream sequences of some genes contain • 5-methylcytosine = Z-like duplex • Structural variations play a role in DNA-protein interactions • RNA-DNA hybrids and ds RNA have an A-type structure

42. Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins H N H N 2 2 The edges of base pairs displayed to DNA major and minor groove contain potential H-bond donors and acceptors: O N n h o h n= Nitrogen hydrogen bond acceptor o= Oxygen hydrogen bond acceptor h= Amino hydrogen bond donor

43. Hydrogen bond donors and acceptors on each edge of a base pair

44. Structural characteristics of DNA facilitating DNA-Protein Recogtnition • Major and major groove of DNA contain sequence- • dependent patterns of H-bond donors and acceptors. • Sequence-dependent duplex structure (A, B, Z, bent • DNA). • Hydrophobic interactions via intercalation. • Ionic interactions with phosphates.

45. Leucine zipper proteins bind DNA major groove Groove binding drugs and proteins 5’-ATT-3’ Others: netropsin, distamycin, Hoechst 33258

46. Triple helix and Antigene approach Hoogsteen base pairing = parallel Reversed Hoogsteen = antiparallel

47. Biophysical properties of DNA • Facile denaturation (melting) and re-association of the duplex • are important for DNA’s biological functions. • In the laboratory, melting can be induced by heating. Single strands T° duplex • Hybridization techniques are based on the affinity of complementary • DNA strands for each other. • Duplex stability is affected by DNA length, % GC base pairs, ionic strength, the presence of organic solvents, pH • Negative charge – can be separated by gel electrophoresis

48. Separation of DNA fragments by gel electrophoresis Polyacrylamide gel: • DNA strands are negatively charged – • migrate towards the anode • Migration time ~ ln (number of base • pairs)