Nucleotides - The 4 S’s

Nucleic Acids A. Nucleotides 1. 2. 3. 4. parts nomenclature numbering properties B. Nucleic Acids 1. Polymer-bond 2. H-bonds 3. Roles a. Nucleotides b. Nucleic acids d. Topology i. Packaging ii. Supercoiling iii.Topoisomerases 4. Stability a. Nucleotides i. Tautomers ii. Acid/base b. Nucleic Acids i. Chemistry ii. Denaturation iii.Stability iv.Nucleases C. The 4 S’s 1. Size a. genomes b. RNAs 2. Solubility 3.Shape a. B-DNA b. A-DNA c. Z-DNA Domain 2 Topoisomerase Type IA Topoisomerase Mechanism Domain 3 Domain 1 How are negative supercoils generated in the first place? 1 Topoisomerase Type II Topoisomerase Mechanism Type II Topoisomerase also called “gyrase” • Tetrameric (a2b2; a=105 kDa, b=95 kDa (ATPase)) • Creates double-stranded breaks • Use of covalent Tyr intermediate • Changes L by –2 • Uses ATP for energy to put in supercoils (DL = –2) Topoisomerase Topoisomerase II Yeast topoisomerase II PDBids 1PVG and 2RGR X-Ray structures of yeast topoisomerase II. Related to Daunorubicin, which inhibits re-joining in (step #5 on previous slide) of Topo II (DNA gyrase) Topoisomerase Inhibitors as Antibiotics/Anticancer Chemotherapeutics 2 The 4 S’s Size Solubility Shape Stability Tautomeric Forms of Bases Uracil (keto) Cytidine (amine) ⥄ Uracil (enol) ⥄ Cytidine (imine) D A N If during replication, an A is in the imino tautomer, if will be complementary to the pyrimidine C rather than T…... H H N A O N N H N N R N N H O H D A N A D R H N H N R A N N A H D N H H N D A O N O D A D T O N R N H N R A D G N N A = ACCEPTOR H D = DONOR N C N R O H A D H N N N R N H O H N N N O R 3 Tautomeric Forms of Bases Mutagenesis Imino tautomer of A * 5’-GGGTTTATTTGGG-3’ 3’-CCCAAATAAACCC-5’ Replication GGGTTTATTTGGG CCCAAATAAACCC * GGGTTTATTTGGG CCCAAACAAACCC Replication GGGTTTATTTGGG CCCAAATAAACCC GGGTTTGTTTGGG CCCAAACAAACCC A*àG G*àA T*àC C*àT Transition mutations are much more common than transversions due to this Acid/base Properties of Bases At pH 7, all bases are neutral. They can fit into middle of B-DNA without repulsions But, acidic or basic treatment will protonate or deprotonate these bases. At low pH, N7 of guanine, N1 of adenine, and N3 of cytosine become protonated. At high pH, N1 of Guanine and N3 of Uracil (Thymine) become deprotonated. 4 Acid/base Properties of Bases Charged bases can de-stabilize the glycosidic bond and mutagenesis pKa 2.4 Destabilizes glycosidic bond, leads to “AP-site” * 7 N O N R N N N H H N H pKa 3.8 H D H N H N N N R H D 1 Leads to A à G mutations A O O N N R O H * “AP-site” refers to a-purinic or a-pyridinic site Other Chemical Changes of Bases DNA Damage: • oxidation • alkylation • degradation 5 Other Chemical Changes of Bases Other Chemical Changes of Bases Deamination of C yields U, which leads to C à T mutations 6 Other Chemical Changes of Bases Frequency 50 In the cell, this deamination is rare, but enough to lead to mutations. In the lab, deamination can be performed readily by treating with nitrous acid 50 2 1 Other Chemical Changes of Bases Point-mutation by Oxidation: Nitrous Acid Deamination of C yields U, which leads to C à T mutations Deamination of A yields Hyp, which leads to A à G mutations 7 Other Chemical Changes of Bases 8-Oxoguanine favors the syn conformation, which leads to O8-G:A bp (G à T mutations) Other Chemical Changes of Bases and other bases 8 Other Chemical Changes of Bases DNA Methylation Some methylations are damaging. • Methylation of G at O6 can basepair with C or T, which causes G à A mutations Some methylations are important. • Methylation for endonuclease restriction recognition or protect DNA from digestion by endonuclease. • Methyl group lie in the major groove and can be used in the interaction with DNA interaction proteins. • Importance of DNA methylation in replication: it is used to differentiate between the new and old strand. If there is a mutation, the repairing system will use the methylated strand as the template. • In mammalian system, the promoter region has regular CpG content. Methylation at these sites can switch off eukaryotic gene expression. Other Chemical Changes of Bases 9 Stability of the Polymers: Nucleic Acids BOTH acid or base lead to ssDNA or ssRNA Acid/base treatment of DNA In Base: deprotonation at G (N1) & U (N3) destabilizes the glycosidic bond, which leads to AP-sites. In Acid: protonates at A (N1), C (N3), & G (N7). For C & G, this also destabilizes the glycosidic bond, which leads to AP-sites AP-sites can lead to cleavage of the phosphodiester bond Acid/base treatment of RNA In acid, similar generation of AP sites Importantly, in Base: complete cleavage of ALL phosphodiester bonds! Stability of the Polymers: Nucleic Acids Base treatment of RNA Base Base Base OH– H2O H+ Base & Nucleoside 2’-phosphate 10 Stability of the Polymers: Nucleic Acids DNA Denaturation Acid or base dsDNA ⇌ ssDNA Acid/base puts charges on bases, which causes internal repulsions and breaks up the double helix This denaturation can also be accomplished using heat. Stability of the Polymers: Nucleic Acids DNA Denaturation UV Spectrum: Native vs. Denatured DNA This denaturation causes a hyperchromic shift. (ssDNA) 40% increase (dsDNA) This makes a nice “observable” (A260) and “perturbable” (heat). What will such a plot of A260 vs. Temp. look like? 11 Stability of the Polymers: Nucleic Acids DNA Denaturation DNA Melting Curve This Tm value is dependent on a number of parameters. The shape is dependent on the sequence and size. Like protein denaturation, this is a cooperative process. Stability of the Polymers: Nucleic Acids DNA Denaturation This Tm value is dependent on: • [salt] • solvents (urea, formamide, guanidine salts) • G:C content of sequence Why? 1x SSC (Salt-Sodium Citrate) In the presence of high concentrations of these solvents, the melting temperatures are much decreased In the presence of high concentrations of salt, the melting temperatures are increased 12 Stability of the Polymers: Nucleic Acids DNA Renaturation ssDNA ⇌ dsDNA •Also called re-annealing or hybridization •Depends on conditions (temp, [salt], solvent) that are maintained BELOW the Tm value. •In addition, the proper formation of the complete, pristine, double helix (completely double-stranded) requires the proper amount of TIME and CONCENTRATION of nucleic acid. •Plots of this are called Cot curves, which are much like Tm curves. •Cot values are dependent on the complexity of the sequence. •Not enough time, you get scrambled structures Partially •Given enough time, very specific annealing occurs…. Renatured DNA Stability of the Polymers: Nucleic Acids DNA Renaturation EXAMPLE: Karyotyping Fluorescence in situ hybridization (FISH) This FISH probe (a fluorescent DNA) found its FISH using fluorescence probe for gene on complementary sequence among 3,000,000,000 bp in each cell! chromosome 21. 13 Stability of the Polymers: What is responsible for STABILITY of dsDNA? C:C nearly as stable as A:U Hoogstein bp is very stable G:G more stable than A:U and as much as G:C Stability of the Polymers: What is responsible for STABILITY of dsDNA What forces operate? If it’s not the H-bonds, then what is it? • Ionic/electrostatics (salt-bridges)? It’s a poly-anion; so charges actually de-stabilize • Hydrophobic? Unlike proteins, where this is the driving force, experiments show that the ssDNA ⇌ dsDNA reaction is enthalpy driven process; ∴ bonds • van der Waals? Yes! This is the most important. As uniform bp come together due to complementarity, the planer bases “stack” on each other and are close enough (<2 Å) to generate induced dipoles. Once started, it “zips” together as long as there are complementary bp being formed. Stacking energy = stability H-bonds in complementary bp = specificity 14 Stability of the Polymers: What is responsible for STABILITY of dsDNA So, if its stacking energy, why are G:C rich sequences more stable than A:T rich sequences? Stacking Energies in B-DNA Adenine ring stacking Stability of the Polymers: Biochemical •Enzymes that catalyze the hydrolysis of the phosphodiester bonds: •These enzymes are called “Nucleases” •Like proteases, if they cleave in the middle, they are called endonucleases (e.g., restriction endonucleases) •If they cleave at the ends, they are called exonucleases Exonucleases can be specific for either 5’-ends or 3’-ends, or either double-stranded or single-stranded nucleic acids (e.g., S1 nuclease) •Also specificity for either DNA (DNases) or RNA (RNAses) •RNAases are very stable, DNAases require Mg+2 cofactor •Can be inhibited by DEPC or EDTA, respectively 15 Nucleotides & Nucleic Acids: preliminary concepts and checkpoints Key Concepts • The nitrogenous bases of nucleotides include two types of purines and three types of pyrimidines. • A nucleotide consists of a nitrogenous base, a ribose or deoxyribose sugar, and one or more phosphate groups. • DNA contains adenine (A), guanine (G), cytosine (C), and thymine (T) deoxyribonucleotides, whereas RNA contains adenine, guanine, cytosine, and uracil (U) ribonucleotides. • Phosphodiester bonds link nucleotide residues in DNA and RNA. • In the DNA double helix, two antiparallel polynucleotide strands wind around each other, interacting in a complementary way via hydrogen bonding between bases in opposite strands, G with C and A with T. (review shape) • RNA molecules are usually single-stranded and may form intramolecular base pairs, G with C and A with U. • DNA carries genetic information in the form of its sequence of nucleotides (see video). • The nucleotide sequence of DNA is transcribed into the nucleotide sequence of messenger RNA, which is then translated into a protein, a sequence of amino acids (see video). The stability of the bases in different tautomer, protonation, deprotonation, and alkylation states leads to mutations. The stability of dsDNA is due to the formation of complementary strands of B-DNA that allows for ideal van der Waals interaction in base stacking. Nucleotides & Nucleic Acids: preliminary concepts and checkpoints Checkpoint • Identify the purines and pyrimidines commonly found in nucleic acids. • Practice drawing the structures of adenine, adenosine, and adenylate • Describe the chemical differences between a ribonucleoside triphosphate and a deoxyribonucleoside monophosphate. • Using Fig. 3-3a as a guide, draw the complete structure of a nucleoside triphosphate before and after it becomes incorporated into a polynucleotide chain. Draw the structure that would result if the newly formed phosphodiester bond were hydrolyzed. • Explain the structural basis for Chargaff’s rules. • Using a three-dimensional computer model of the DNA molecule, identify each of the following structural features: the 3‘ and 5’ end of each strand, the atoms that make up the sugar–phosphate backbone, the major and minor grooves, the bases in several base pairs, and the atoms that participate in hydrogen bonding in A·T and G·C base pairs. • List the structural differences between DNA and RNA. 16