Biochemistry of Medicinals I – Nucleic Acids Instructor: Natalia Tretyakova, Ph.D. 760E CCRB (Cancer Center) Tel Lecture: MWF 3:35-4: WDH Recitation: Th Web page: see “Web enhanced courses”

Chapter 1. DNA Structure. Required reading: Stryer 5 th Edition p , , 152, , , ) (or Stryer’s Biochemistry 4 th edition p ,80-88, , , , )

DNA Structure: Chapter outline 1.Biological roles of DNA. Flow of genetic information. 2.Primary and secondary structure of DNA. 3.Types of DNA double helix. Sequence- specific DNA recognition by proteins. 4.Biophysical properties of DNA. 5.DNA topology. Topoisomerases. 6.Restriction Endonucleases. Molecular Cloning

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

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

The Building Blocks of DNA  -N-glycosidic bond

DNA and RNA nucleobases (DNA only) (RNA only)

Purine Nucleotides

Pyrimidine Nucleotides

Adenine Example: nucleobase

2’-deoxyadenosine Nucleoside

2’-deoxyadenosine-5’-monophosphate Nucleotide

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

Structural differences between DNA and RNA DNA RNA

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

Nucleosides Must Be Converted to 5’-Triphosphates to be Part of DNA and RNA

DNA is Arranged 5’ to 3’ Connected by Phosphates Linking in DNA biopolymer: DNA primary structure

DNA secondary structure – double helix James Watson and Francis Crick, 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 CrickJim Watson

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

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

Living Figure – B-DNA

DNA is Composed of Complementary Strands

Base Pairing is Determined by Hydrogen Bonding same size

Base stacking: an axial view of B-DNA

Forces stabilizing DNA double helix 1.Hydrogen bonding (2-3 kcal/mol per base pair) 2.Stacking (hydrophobic) interactions (4-15 kcal/mol per base pair) 3. Electrostatic forces.

right handed helix planes of bases are nearly perpendicular to the helix axis. 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 B-DNA 3.4 A rise between base pairs Wide and deep Narrow and deep 7.0 A helical axis passes through base pairs 23.7 A

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

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

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

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

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

Living Figure – A-DNA

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

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

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

Z-form double helix: polynucleotides of alternating purines and pyrimidines (GCGCGCGC) at high salt Left handed helix 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 planes of the bases are tilted 9° relative the helix axis. Flat major groove Narrow and deep minor groove 18.4 A 3.8 A rise between base pairs

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

Living Figure – Z-DNA

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 RNA-DNA hybrids and ds RNA have an A-type structure Structural variations play a role in DNA-protein interactions

Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins N H 2 NO H 2 N hnho n= Nitrogen hydrogen bond acceptor o= Oxygen hydrogen bond acceptor h= Amino hydrogen bond donor The edges of base pairs displayed to DNA major and minor groove contain potential H-bond donors and acceptors:

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

Structural characteristics of DNA facilitating DNA-Protein Recogtnition 1.Major and major groove of DNA contain sequence- dependent patterns of H-bond donors and acceptors. 2.Sequence-dependent duplex structure (A, B, Z, bent DNA). 3.Hydrophobic interactions via intercalation. 4.Ionic interactions with phosphates.

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

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

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. 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 T°T° Single strands duplex

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