DNA is Composed of Complementary Strands

1. DNA is Composed of Complementary Strands

2. Base Pairing is Determined by Hydrogen Bonding same size

3. 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.

4. B-DNA • •Sugars are in the 2’ endo conformation. • •Bases are the anti conformation. • •Bases have a helical twist of 36º • (10.4 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

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

6. 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

7. 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’

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

9. 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

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

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

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

13. 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

14. 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

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

17. 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

18. 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

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

20. 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.

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

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

23. 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

24. 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)

25. DNA Topology DNA has to be coiled to fit inside the cell DNA polymers must be folded to fit into the cell or nucleus (tertiary structure).

26. DNA Topology:

27. DNA Topology: linking number

28. Consider a 260 bp B-duplex:

29. Connect the ends to make a circular DNA: Tw = 260/10.4 = 25

33. An electron micrograph of negatively supercoiled and relaxed DNA Stryer Fig. 27.20

34. Organization of chromosomal DNA • Chromosomal DNA is organized in loops (no free ends) • It is negatively supercoiled: 1 (-) supercoil per 200 nucleotides 145 bp duplex Histone octamer (H2A, H2B, H3, H4)2 H1 is bound to the linker region

35. Enzymes that control DNA supercoiling: DNA Topoisomerases Change the linking number (Lk) of DNA duplex by concerted breakage and re-joining DNA strands Topoisomerase enzymes Topoisomerases I Relax DNA supercoiling by increments of 1 (cleave one strand) Topoisomerases II Change DNA supercoiling by the increments of 2 (break both strands) Usually introduce negative supercoiling

36. Human DNA Topoisomerase I: DNA: side view 20Å Stryer Fig. 27.21

37. Mechanism of DNA Topoisomerases I 723 OH P-Topo  Wr = 1

38. Drugs that inhibit DNA Topoisomerase I • • Camptothecin, topotecan and analogs • •Antitumor activity correlates with interference with topoisomerase activity • • Stabilizes topoisomerase I-DNA intermediate, preventing DNA strand re-ligation • Used in treatment of colorectal, ovarian, and small cell lung tumors

39. Enzymes that control DNA supercoiling: DNA Topoisomerases Change the linking number (Lk) of DNA duplex by concerted breakage and re-joining DNA strands Topoisomerase enzymes Topoisomerases I Relax DNA supercoiling by increments of 1 (cleave one strand) Topoisomerases II Change DNA supercoiling by the increments of 2 (break both strands) Usually introduce negative supercoiling

40. Topoisomerases II • Most of Topoisomerases II introduce negative supercoils (e.g. E. coli DNA Gyrase) • Require energy (ATP) • Each round introduces two supercoils ( Wr = - 2) • Necessary for DNA synthesis • Form a covalent DNA-protein complex similar to Topoisomerases I

41. Yeast DNA Topoisomerase II Stryer Fig. 27.23

42. Topoisomerase II - mechanism Stryer Fig. 27.24

43. Drugs that inhibit bacterial Topoisomerase II (DNA gyrase) Interfere with breakage and rejoining DNA ends: Inhibit ATP binding:

44. Enzymes that cut DNA: exonucleases Phosphate group Nucleobase 2’-deoxyribose HO 5’ A OH 3’ 5’ 3’ 5’ + dNMPs • Degrade DNA in a stepwise manner by removing deoxynucleotides in 5’  3’ (A) or 3’  5’ direction (B) • Require a free OH • Most exonucleases are active on both single- and double-stranded DNA • Used for degrading foreign DNA and in proofreading during DNA synthesis B HO H 3’

45. DNA Endonucleases • Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate ends 5’ 3’-OH 5’-P 5’-P 3’-OH • some endonucleases cleave randomly (DNase I, II) • Type II Restriction endonucleases are highly sequence specific EcoRI recognition site: Palindromic site (inverted repeat) • RE are found in bacteria where they are used for protection against foreign DNA

46. Recognition sequences of some common restriction endonucleases

47. DNARestrictionEnzyme EcoR V

48. Applications of Restriction Endonucleases in Molecular Biology • DNA fingerprinting (restriction fragment length polymorphism). • 2. Molecular cloning (isolation and amplification of genes).

49. Southern blotting

50. Restriction fragment length polymorphisms are used to compare DNA from different sources