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Virginia Tech BCHM 4116 - Tertiary Structure and Quantification of DNA

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BCHM 4116 1st Edition Lecture 3 Outline of Last Lecture I. Structural basis of genetic material: Nucleic acidsa. DNA vs. RNAII. Purine and Pyrimidine Basesa. Keto-enol tautomeric shiftsIII. DNA better suited as genetic material Outline of Current Lecture I. Watson and Crick base pairing II. What is DNA helix stabilized byIII. 3 types of DNAIV. Biological Implications of double stranded helix in DNAV. Intercalating Agents VI. Structural Transitions of DNAVII. Hyperchromic shift VIII. DNA structure is dynamicIX. Tertiary structure of DNAX. L= T+WXI. Organization chromatin and chromosome Current LectureWatson and Crick base pairingWatson and Crick determined four physical properties of DNA 1. Antiparallel strandsa. One strand of the DNA flows from 5’ to 3’, whereas the complimentary strand goes from 3’ to 5’. 2. Interchain Hydrogen bonds form Watson-Crick base pairsa. Hydrogen bonding occurs between base pairs of the two strands. b. The base pairs are:i. Guanine pairs with cytosineii. Adenine pairs with thymine3. Diameter of the helical width and inter-base stacking distance a. 2 nanometers (helical width)b. 0.34 nanometers (inter-base stacking distance)These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.4. There are 10 base pairs per repeat What is DNA helix stabilized byThere are 3 main forces that play a part in stabilizing the DNA helix1. Hydrophobic and van der Waals between stacked basesa. The base pairs stack together through pi, pi electronic interactions and hydrophobic forces b. Major groove/minor groove: i. Formed by the unequally spaced sugar-P back bone of the helixii. Some proteins can recognize H-bonding possibly in these grooves2. Hydrogen bonding between base pairsa. This is an interchain bond that holds the complementary bases together3. Phosphate with watera. Hydrogen bonds between base pairs are replaced with hydrogen bonds between individual bases and water molecules when the two strands are separatedb. Additionally, polar atoms in the sugar-P backbone of DNA form hydrogen bonds with water molecules 3 types of DNAThere are three main forms of double helical structuresA-DNA B-DNA Z-DNARight handed Right handed Left handedDNA normally assumes this structureBiological implications of double stranded helix in DNA1. Complementary strands faithful replicationa. Since both strands of a DNA molecule are identical, their use as a template for new DNA ensure identical replicates 2. Genetic information can be stored as a unique base sequencesa. 3 bases make up one codon, which is translated into one protein. There are 4 choices for each base in a 3-base codon, 4x4x4=64 choices! b. So many possibilities allows for unique base sequences3. Disassociation and re-association can be relatively easily achieveda. Because bases only associate with their complement, reassociation after denaturation is much like zipping up a zipper4. The existence of major and minor groovesa. These exist as recognizable sites for certain proteins/enzymesIntercalating agentsThere are three main intecalating agents. These bind to DNA, inserting themselves between stacked base pairs, and fluoresce, allowing us to see the molecule. The three intercalating agents are ethidium bromide, acridine orange, and actinomycin D. Structural transitions of DNAThe second structure represents denatured DNA, and the last structure represents the renatured DNA. Hyperchromic shiftHyperchromic shif is defined as the increase in the absorbance of DNA upon denaturation. Tm: melting temperature, the midpoint of the hyperchromic shiftThere are several factors that affect Tm1. GC contenta. More stacking energy2. Salt concentrationa. Salt neutralizes phosphate, creating a more stable structure3. Hydrogen bondinga. Destabilizes DNA, bonds with bases so the bases don’t interact with each-other 4. Extreme pH a. Denatures DNADNA structure is dynamicDNA sequences that are inverted repeats, or palindromes, have the potential to form a tertiary structure known as a cruciform if the normal interstrand base pairing Is replaced by intrastrand pairing Tertiary structure of DNAIn double stranded DNA, there are two strands are wound around one another every 10 base pairs. Double stranded DNA forms supercoils. Supercoils form when strands are underwound (negative) or overwound (positive). Negative supercoils are relaxed between twists, where as positive supercoils are tight around the twists. Nucleomatrix: structure DNA is attached to at times when supercoiled. L= T+WLinking number (L): the number of time that the 2 strands are intertwined. L does not change unless the covalent bond is in at least one of the strands is broken.Twist (T): the number of helical turnsWrithe (W): the number of supercoilsLinking number = twist + writheThe linking number can be changed by only break one or both of the DNA strands  windingthem tighter or looser and rejoining the ends. Topoisomerases are a family of enzymes capable of doing just this. Topoisomerase I cuts one strand of DNA. Topoisomerase II cuts both strands of DNADNA Gyrase: the bacterial enzyme is a topoisomerase that introduces negative supercoils into DNASuperhelix Density: the difference between the linking number of a DNA and the linking numberof its relaxed form is (L-L0)/L0L0 is the linking numer of relaxed DNA Organization chromatin and chromosomeHistones are proteins that DNA wraps itself around when in chromosome form. Genes are regulated through histone acetylation (much like methylation with hyperchromic shift) and chromatin


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