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Transformative Advances in Molecular Biology Dr. Mark Settles and Dr. Kevin Folta Horticultural Sciences Department and the Graduate Program in Plant Molecular and Cellular Biology University of Florida Gainesville, FL 32611 http://pmcb.ifas.ufl.edu amsettles@ifas.ufl.edu kfolta@ifas.ufl.edu

DNA Structure : 

DNA Structure by Mohamad Fadhli Mad Atari Brent O’Brien 2

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DNA Structure Experimental Evidence (X-ray crystallography) - Stokes, A. R., and Wilson, H. R. (1953) Molecular Structure of Deoxypentose Nucleic Acids. - Franklin, R. E., and Gosling, R. G. (1953) Molecular Configuration of Sodium Thymonucleate. Structural Model and Genetic Implications - Watson, J. D., and Crick, F.H. C. (1953) A Structure for Deoxyribose Nucleic Acids. - Watson, J. D., and Crick, F.H. C. (1953) Genetic Implications of the Structure of Deoxyribonucleic Acid. All papers published in Nature 3

Molecular Structure of Deoxypentose Nucleic Acids. Stokes, A. R., and Wilson, H. R. (1953) : 

Molecular Structure of Deoxypentose Nucleic Acids. Stokes, A. R., and Wilson, H. R. (1953) Provides experimental evidence for the polynucleotide chain being helical This is the natural configuration This structure is the same in all species Base ratios differ across species Polynucleotide chains may assemble in crystalline, semi-crystalline, and paracrystalline forms All forms show two distinct regions in X-ray diffraction photos One region is due to regular spacing of nucleotides on the chain The other is due to longer spacings of chain configuration 4

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The absence of reflection at the meridian suggests a helical structure The strong 3.4 A. reflection is due to inter-nucleotide repeat along the axis The 34 A. layer lines are due to repeats of chain configuration 5

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Diffraction by Helices The intensity distribution in the diffraction pattern of a series of points equally spaced along a helix is given by the squares of Bessel functions A uniform, continuous helix gives a series of layer lines of spacing corresponding to the helix pitch A straight line may be drawn through the innermost maxima of each Bessel function and the origin the angle this line makes with the equator is roughly equal to the angle between an element of the helix and the helix axis 6

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Interpretation of the X-ray photograph Layer-line width suggests intensities correspond more closely to a single helix, indicating that the dominant helix has a pitch of ~34 A. The angle of the helix indicates that its diameter is ~20 A. Two or three intertwined, coaxial helices ( with 10 nucleotides per turn) would be required to obtain a reasonable number of nucleotides per unit length of fibre 7 5th layer

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Interpretation of the X-ray photograph The absence of reflection on or near the meridian (AAA ) is a direct consequence of helical structure The empty region near the equator (BBB) is a result of radial distribution of nucleotide shape 8

Structure in vivo; an emerging pattern : 

Structure in vivo; an emerging pattern X-ray spectra from sperm heads and trout semen is determined by the helical form-function X-ray data from bacteriophage show the main features of paracrystalline sodium nucleate Active deoxypentose nucleate has the same crystalline structure as sodium thymonucleate 9

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Sodium thymonucleate- A form (75% relative humidity) and B form (higher humidity). AB (water content;40-50% change), reversible. B forms- sheath of water of each units of sodium thymonucleate. Free from neighboring molecules influence Molecular Configuration in Sodium Thymonucleate Franklin, R. E., and Gosling, R. G. (1953) 10

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The fiber axis period is 34 A. + strong reflection at 3.4 A. = suggests 10 residues per turn 34 A 3.4 A 11

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Radius measurements value of 10 A. =20A. Diameter r =10 A 20 A 12

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Phosphorous must lie on the outside of the helix, as the linear array of maxima are one of the diagram’s strongest features At a 10 A. radius P atoms would be 7.1 A. apart which corresponds to the expected distance in a fully extended molecule Sugar and base groups must face the helical axis P SUGAR BASE 7.1 A P SUGAR BASE 13 P P P P P P P P P P P P P P P S B S B

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A cylinder of radius 10 A. and height 34 A. would contain 32 nucleotides. One repeating unit will contain 10 nucleotides on each of two or on each of three co-axial molecules. 32 nucleotides 32 nucleotides 14 10 nucleotides

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Previous models Pauling and Corey proposed; - a triple helix -phosphates near the fiber axis - bases on the outside Fraser proposed; - a triple helix -phosphates on the outside - bases linked by H-bonds near the axis Structural Model Watson, J. D., and Crick, F.H. C. (1953) A Structure for Deoxyribose Nucleic Acids. 15

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Structural Model and Genetic Implications - Watson, J. D., and Crick, F.H. C. (1953) A Structure for Deoxyribose Nucleic Acids. - Watson, J. D., and Crick, F.H. C. (1953) Genetic Implications of the Structure of Deoxyribonucleic Acid. Structural Model Two right-handed helical chains coiled around a common axis 3’ to 5’ linkages The chains are related by dyads perpendicular to the helix axis Helix chains are anti-parallel Sugars are perpendicular to bases Nucleotides are separated by 3.4 A. in th z-direction There is a 36° angle between adjacent bases The structure repeats every 34 A., or 10 residues The helix diameter is 20 A. The structure is open with high water content Held together by H-bonding of purine and pyridimine bases 16

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Base-pairing Model A single base from one chain H-bonds to a single base from the other chain One base must be a purine and the other must be a pyridimine (due to conformational restrictions) H-bonds are made at purine position 1 to pyrimidine position 1, and purine position 6 to pyrimidine position 6 If bases exist in their most plausible tautomeric forms then only two pairs can occur Adenine + Thymine Guanine + Cytosine The ratios of A to T and G to C have been shown to be close to unity The sequence of one chain is complementary to the other The sequence of bases on a single chain is not restricted 17

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Genetic Implications of the proposed DNA structure Considering a DNA model that is complimentary, anti-parallel, and not restricted in sequence of bases, Watson and Crick hypothesize that; A long DNA molecule could have many permutations and the sequence of bases code for genetic information DNA is a pair of templates, one complimentary to the other Specific events must occur during replication 18

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Requirements for replication Uncoiling/de-condensation H-bonds are broken The chains unwind and separate Free nucleotides H-bond to the template strand Each chain serves as a template to generate two pairs of identical chains Stearic properties of the bases dictate that this method of replication is only possible if the resulting chain takes on the proposed structure The authors question whether an enzyme is required 19

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The role of a groove-occupying polypeptide chain The authors speculate that there is room for a polypeptide chain to occupy the space between the pair of polynucleotide chains. The 7.1 A. between P atoms is close to the repeat of a fully extended polypeptide The weakness of the 2nd layer-line of X-ray diagrams is “crudely” compatible with this idea Proposed functions of the polypeptide chain are; Control of coiling and uncoiling Holding a single DNA strand in helical conformation 20 P SUGAR BASE 7.1 A P SUGAR BASE 2nd layer

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Thank You for Watching! Other topics in this series: DNA as the hereditary material Structure of DNA Universal amino acid code DNA Sequencing technology Restriction enzymes Polymerase Chain Reaction Transposons RNA splicing Mechanism of translation Mechanisms of RNAi PMCB Interested in Plant Molecular and Cellular Biology? The interdisciplinary PMCB group at the University of Florida is always welcoming qualified graduate students! An undergraduate major in Plant Molecular and Cellular Biology is offered through the Horticultural Sciences Department