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Biochemistry of Medicinals I – Nucleic Acids

This lecture explores the primary and secondary structure of DNA, types of DNA double helix, sequence-specific DNA recognition by proteins, biophysical properties of DNA, DNA topology, and restriction endonucleases. It also discusses the relevance of DNA structure in various fields such as cancer, genetic diseases, genetic typing, rational drug design, and biotechnology.

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Biochemistry of Medicinals I – Nucleic Acids

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  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 2:30-3:20 7-135 WDH Web page: see “Web enhanced courses”

  2. Chapter 1. DNA Structure. Required reading: Stryer 5th Edition p. 117-125, 144-146, 152, 745-750, 754-762, 875-877) (or Stryer’s Biochemistry 4th edition p. 75-77,80-88, 119-122, 126-128, 787-799, 975-980)

  3. 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

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

  5. 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

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

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

  8. Purine Nucleotides

  9. Pyrimidine Nucleotides

  10. 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

  11. Structural differences between DNA and RNA DNA RNA

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

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

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

  15. 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

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

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

  18. DNA is Composed of Complementary Strands

  19. Base Pairing is Determined by Hydrogen Bonding same size

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

  21. 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.

  22. 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

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

  24. 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’

  25. 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

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

  27. 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

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

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

  30. 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

  31. 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

  32. 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

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