Office Hours start today at 2:30-4:30pm Jon Paczkowski Weill 367 Review on Monday Feb 2, WRN245 Adam & Patrice Absorption spectra of the common nucleotides Measurement for NA concentrations Ch8:8 (9) High energy bonds a ? ? p.10 Nucleoside 5'-triphosphates are carriers of energy ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism Bases serve as recognition units Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction Functions of Nucleotides 5? 3? DNA and RNA chains have polarity Follow the sugar-P backbone to determine polarity Shorthand notations for oligonucleotide chains 3? 5? P 3? 5? G P 3? 5? C P 3? 5? A P 3? 5? T P OH T P G C A T T 5? 3? or simply Study guide p.107, #1, 2, 4, 7- 9 Chapter 8: 3, 8, 10 5? TGCAT 5 C sugar Chemistry of nucleic acids The double helix Movie:2902 The double helix 10-01 Lehninger 3rd P = yellow, C= black, H= white, N=blue, O= red Chapter 8: 6, 9, 12 (6, 13) Study guide, p.108, #5, 10, 13-14 DNA and RNA have many similarities, but also differences: 1. DNA is usually double-stranded, and therefore rigid RNA is single-stranded and flexible 2. The nucleotides of DNA are ATGC, RNA are AUGC. 3. RNA has 2?OH, they are susceptible to alkali hydrolysis while DNA is not. 4. N-glycosidic bonds of purine bases of DNA are sensitive to acid hydrolysis but not RNA deoxyribose ribose Spontaneous deamination of cytosines results in mutations. Why is it important that DNA contains thymine not uracil? Also see figure 8-33 C:G G:C U:A G:U replication mutation G:C C:G C:G repair replic Uracil N- glycosylase Thymine Spontaneous deamination of cytosines results in mutations. G:C to G:U in DNA, U will be recognized as a mutation because Us are not naturally found in DNA. If DNA contains U instead of T just like RNA, then there is no distinction between products of cytidine deamination and the endogenous Us in DNA. Why is it important that DNA contains thymine not uracil? In mammalian cells, deamination of cytosine to uracil occurs about one of every 107 cytidine residues in 24 hours, which corresponds to about 100 spontaneous events per cell per day. Hydrolysis of Nucleic Acids DNA is not susceptible to base (alkali) RNA (phosphodiester bond) is hydrolyzed by dilute base RNA is resistant to dilute acid DNA is depurinated by dilute acid. 1mM HCl leads to hydrolysis of purine glycosidic bonds and the loss of purine bases from the DNA Enzymatic hydrolysis of NA by nucleases Nucleases are enzymes that cleave phosphodiester bonds Figure 8-8 3? 2? Alkali hydrolysis of RNA 3? 2? 5? Depurination of DNA by acid 1? 9 Nucleases have specificity - may cleave at the 5? or 3? side of the phosphodiester bond Convention: The 3? side of each 5C sugar is termed a; the 5? side is termed b Hydrolysis of the a bond yields 5?-PO4 products Hydrolysis of the b bond yields 3?-PO4 products Mixture of 5?-nucleoside monophosphate (NMPs) A 3?-5?-diPO4 nucleoside from the 5?-end A mixture of 3? NMPs A nucleoside from the 3?-OH end Nucleases that cleave from the termini of polynucleotide chains are exonucleases if they cleave from the 3? termini they are 3?-5? exonucleases Nucleases that cleave internal phosphodiester bonds are endonucleases Nucleases that cleave RNA are called Rnases (ribnucleases) Nucleases that cleave DNA are called Dnases (deoxyribonucleases) Restriction nucleases are endonucleases that cleave at a specific nucleotide sequence by recognizing specific base sequences Specificity of Various Nucleases Enzyme DNA, RNA a or b Specificity or both Exonuclease Snake venom phosphodiesterase Both a 3? to 5?, 5? NMP products Spleen phosphodiesterase Both b 5? to 3?, 3? NMP products Endonucleases Rnase A (pancreas) RNA b 3?-P to Py, oligos with 3?-P B. subtilis Rnase RNA b 3?-P to Pu, oligos with 3?-P Rnase T1 RNA b 3?-P to G Rnase T2 RNA b 3?-P to A Dnase I DNA a between Py and Pu, nicks ds DNA Dnase II DNA b oligo products Nuclease S1 Both a cleaves ss but not ds NA Three dimensional structure of DNA http://www.sdsc.edu/ScienceWomen/franklin.html 1. X-ray fiber diffraction data from Rosalind Franklin and Maurice Wilkins 2 periodicities: an internucleotide distance of 3.4 Å 1 complete turn every 34Å or 10 nucleotides/turn The three-dimensional structure of DNA The Watson and Crick DNA Double Helix - 1953 Solved structure by model building based on: 2. Chargaff?s Rule on base composition of DNA (late 1940?s) [A] = [T], [G] = [C]; [A]/[T] = [G]/[C] = 1 [A+G] = [T+C], or [Pu] = [Py] This rule applies to DNA in all species studied Figure 8-14 Cross indicates helical structure From density of DNA, diameter, length of helical turn (34Å), and 10 nuc/turn, deduced that helix must contain 2 polynucleotide chains D=M/V or M=D.V Diameter = 20 Å 10.5 bp /turn Sugar-P backbone is perpendicular to the planar base pairs 5? 5? 3? 3? Pitch length The Watson-Crick B form DNA 3. Deduced by model building 11Å 20Å Fig 8-15 charged PO4-- on the outside Hydrophobic bases on the inside Minor groove Major groove 2 H bonds 3 H bonds major minor 2 6 4 4 5 6 http://www.callutheran.edu/BioDev/omm/jmol/dna_intro/start.html#top Top view C1? C1? The two strands of the helix have complementary notations that can serve as templates for the synthesis of new DNA strands Structure informs function
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