MHS CMB Class I PDF

INTRODUCTION Instructor Minu Chaudhuri, Ph.D. Professor Department of Microbiology and Immunology Email: [email protected] Phone: 615 327 5726 Office: West Basic Science Bldg. Room # 4105 DNA Structure and Function Suggested Textbooks • Molecular Biology of the Cell. Alberts et al., 6th edition...

INTRODUCTION Instructor Minu Chaudhuri, Ph.D. Professor Department of Microbiology and Immunology Email: [email protected] Phone: 615 327 5726 Office: West Basic Science Bldg. Room # 4105 DNA Structure and Function Suggested Textbooks • Molecular Biology of the Cell. Alberts et al., 6th edition, Chapter 4 Objectives After this class students will learn; u The chemical structure of DNA, the components of nucleotides, how to number the pentose sugar u To differentiate nucleotide from nucleoside, different types of chemical bonds in the structure of nucleotide u Structures of the purines and pyrimidine, u Discovery of DNA structure u to explain the meaning of anti-parallelism of DNA strands and base pairing u how 5’ and 3’ ends of DNAs are formed u different forms of DNA double helices u to differentiate palindromic and mirror repeats u RNA structure, different types of RNAs and their functions u Chromosome structure, differences between chromatin and chromatid Central Dogma of Genetics Information flow in the biological system Major Biopolymers Polymer Monomer DNA Deoxyribonucleotide RNA Ribonucleotide Protein Amino acid Building blocks of DNA DNA molecule – composed of 4 deoxyribonucleotides (deoxyribose, nitrogenous base, phosphate) Deoxyadenosine monophosphate Deoxycytidine monophosphate Deoxyguanosine monophosphate Deoxythymidine monophosphate (dAMP) (dCMP) (dGMP) (dTMP) Nucleoside VS Nucleotide Ester bond N-glycosidic bond • Nucleosides are base plus sugar • Nucleotides are nucleoside plus phosphate • May be 1 to 3 phosphates Abbreviation and nomenclature of the building blocks of DNA and RNA Base Nucleoside Nucleotide Adenine (A) Adenosine (A-R) Adenylic acid (A-R-P) Guanine (G) Guanosine (G-R) Guanylic acid (G-R-P) Cytosine (C) Cytidine (C-R) Cytidylic acid (C-R-P) Thymine (T) Thymidine (T-R) Thymidylic acid (T-R-P) Uracil (U) Uridine (U-R) Uridylic acid (U-R-P) Purine VS Pyrimidine 5 8 7 9 4 6 1 2 3 3 4 5 2 1 6 Chemical Bonds • Bases connect to sugar by N-glycosidic bonds • 1’ C of sugar to N1 of pyrimidine or N9 of purine • Phosphate attached to 5’ carbon of sugar • New bases added at 3’ carbon, need OH dATP dCTP 5’ 9 5’ 1’ 3’ 1 1’ 3’ Base modifications • • • • Methylation Hydroxymethylation Glycosylation Acetylation DNA is the genetic material Avery-MacLeod-McCarty Experiment Note: In 1928, Frederick Griffith performed this experiment and discovered the transformation process. Later in 1944 Avery et al., revisited the same experiment in a more definitive way and showed that DNA is the genetic material that causes this transformation. Chargaff’s Rule In 1940, Erwin Chargaff and his colleagues concluded the following facts about DNA The Key to Crack the DNA Structure In 1950, Maurice Willkins, and Rosalind Franklin generated the X-ray picture of DNA fiber In 1953, Jim Watson, Francis Crick solved the DNA structure Photo 51 The Double Helical Structure of DNA • The DNA molecule has a double helix shape similar to a spiral staircase • The handrails are made of sugar and phosphate • The steps of the ladder: • A always pairs with T • G always pairs with C Double Helix Stabilization • Hydrogen bonds between complementary bases • Contribute about 15% of the stability • A-T base pairs have 2 H bonds • G-C base pairs have 3 H bonds - ~50% stronger than A-T • Base stacking interactions • Contribute 85% of stability • Van der Waals and hydrophobic interactions between adjacent base pairs Minor and Major Grooves Direction of DNA Strands are Antiparallel • Strands have a direction set by the backbone • Strands are synthesized from a 5’ to 3’ direction • Paired strands run in opposite directions • Necessary for hydrogen bonding to hold strands together 3’ and 5’ Ends of DNA strand DNA Synthesis Requires a Free 3’ OH Group B-Form of DNA Helix • Right-handed helix • There are 10.5 bases per turn; • 3.4 angstroms between stacked bases • 36 angstroms per helical turn Anomeric Forms of Nucleotides Syn VS Anti Left-handed helix will turn counterclockwise Right-handed helix will turn clockwise α vs β anomer Different Forms of DNA Double Helix • B-DNA is most common; right-handed helix • A-type helix known in DNA-RNA hybrids; right-handed helix, most compact • Z-DNA seen in GC-rich regions; left-handed helix Denaturation of DNA Strands • Denaturation means separation of strands by breaking hydrogen bonds by: • • • • pH changes- low or high Solvents Formamide Increasing temperature • DNA strands separate • Base composition determines denaturation temperature – A-T pair denatures at lower temperature, only two bonds vs. 3 for GC pair • Can be monitored by UV absorption – Bases absorb at 260 nm, absorb more when more exposed in single strands DNA Absorbance Spectra Single VS Double Stranded DNA Melting Temperature [G+C] = 35% 50% 66% • Possible under specific conditions • Repetitive sequences can renature more quickly than unique sequences • Important for experimental techniques Palindrome and Mirror Repeats Restriction endonuclease and many DNA-binding sequence recognize such repeats Hairpin and Cruciform structure of DNA Inverted repeats in the same strand could form hairpin structure Inverted repeats in both strands could form cruciform structure Triplex and Tetraplex DNA Structure § Triplex structure could be formed in GC rich region. Examples: telomeric cap § Highly stable § A synthetic DNA strand designed to make triple helix with a gene regulatory region could disrupt gene expression. § This strategy has been exploited to develop medicine § Tetraplex structure could be found in high G rich region Procaryotic VS Eukaryotic Chromosomes Bacteria Animal cell Replication One DNA molecule Nucleotides (ATP, GTP, CTP, TTP) and DNA Polymerase Two DNA molecules and Pyrophosphate Three postulated methods of DNA replication Semi-conservative Replication of DNA Messelson-Stahl Experiment Problem-1 Spontaneous deamination of certain bases in DNA occurs at a constant rate under all conditions. Such deamination can lead to mutations if not repaired. Which deamination indicated below would lead to a mutation in a resulting protein if not repaired? (A) T to U (B) C to U (C) G to A (D) A to G (E) U to C DNA vs RNA Property Sugar Distinctive base Stability to NaOH Polymer is usually: Usual # bases DNA Deoxyribose Thymine (T) Stable Double-stranded Millions Thymine RNA Ribose Uracil (U) Labile Single-stranded 100-10,000 Uracil Only in RNA! Many Types of RNA • RNA has many functions, different forms for each function • Generally single-stranded • Three major functional forms • rRNA, structural and functional components of ribosomes, majority of RNA in most cells • mRNA, carries protein coding sequence • tRNA, matches amino acid with codon Basics of mRNA Structure • Not all of mRNA codes for protein • 5’ end has leader sequence and protective cap structure • 3’ end has untranslated region and poly-A tail • Only coding region is translated into protein tRNA structure Non-coding RNAs § Only a small fraction of DNA codes for proteins, and a very small fraction of the non-protein-coding DNA consists of genes for RNA such as rRNA and tRNA § A significant amount of the genome may be transcribed into noncoding RNAs (ncRNAs) § There are various types of ncRNAs. These include a) small nuclear RNA (snRNA, ~150 b) b) long nuclear RNA (lncRNA, >200 b) c) small nucleolar RNA (snoRNA) d) microRNA (miRNA) e) small interfering RNA (siRNA) f) piwi-interacting RNA (piRNA) § ncRNA performs various functions, e.g., 1. splicing 2. mRNA degradation, 3. epigenetic modification, 4. genomic imprinting, 5. X-chromosome inactivation, 5. telomere protection Problem-2 A 32-year-old woman exhibited a high fever, malaise, generalized lymphadenopathy, weight loss, and esophageal candidiasis. She had a history of drug abuse and needle sharing. Blood analysis indicated a CD4 lymphocyte count of less than 200. Which of the following compounds would be a drug of choice for this patient? (A) Adenosine (B) Methotrexate (C) Deoxy adenosine (D) Dideoxy adenosine (E) 5-Flurouracil