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Dna lecture

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  • 1. Deoxyribonucleic Acid
    (DNA)
  • 2.
  • 3. The double helix
  • 4. Nitrogenous Bases and Pentose Sugars
  • 5. Purine and Pyrimidine Structure
    (1)  Pyrimidines are planar
    (2)  Purines are nearly planar
    (3) Numbering is different
  • 6. Numbering Is Different
  • 7.
  • 8. Bases Have Tautomeric Forms
    Uracil
  • 9.
  • 10. Glycosidic bond
    Nucleosides vs. Nucleotides
  • 11. Nucleotides formed by condensation reactions
  • 12.
  • 13. Monophosphates
  • 14. Deoxyribonucleotides
  • 15. Ribonucleotides
  • 16. Only RNA Is Hydrolyzed by Base
  • 17. Nucleoside Diphosphate and Triphosphate
  • 18. Ester bonds
    Dinucleotides and Polynucleotides
  • 19. G=C
    Watson-Crick Base Pairs
    A=T
  • 20. Hoogsteen Base Pairs
  • 21. Other Base Pairs Are Possible
    Watson-Crick,
    Reverse Watson-Crick,
    Hoogsteen,
    Reverse Hoogsteen,
    Wobble,
    Reverse Wobble
    Homo Purines
    Hetero Purines
  • 22. Base Pairing Can Result in Alternative DNA Structures
    Triplex
    Tetraplex
    Cruciform
    Hairpin Loop
  • 23. Periodicity: A pair of strong vertical arcs (C & N atoms) indicate a very regular periodicity of 3.4 Å along the axis of the DNA fiber.
    Astbury suggested that bases were stacked on top of each other "like a pile of pennies".
    Helical nature: Cross pattern of electron density indicates DNA helix and angles show how tightly it is wound.
    Diameter: lateral scattering from electron dense P & O atoms.
  • 24. DNase can only cleave external bond demonstrating periodicity
  • 25. Watson and Crick Model (1953)
    Hydrophilic
    Hydrophobic
    Complementarity
    2 long polynucleotide chains coiled around a central axis
    Bases are 3.4 Å (0.34 nm) apart on inside of helix
    Bases flat & lie perpendicular to the axis
    Complete turn = 34 Å
    10 bases/turn
    Diameter = 20 Å
    Alternating major and minor grooves
  • 26. Base Pairing Results from H-Bonds
    Only A=T and GC yield 20 Å Diameter
  • 27. A:C base pair incompatibility
  • 28. Bases Are Flat
  • 29. Chains Are Antiparallel…
  • 30. …Because of Base Pair Torsional Bond Angles
  • 31. Base Pairs and Groove Formation
  • 32. Base flipping can occur
  • 33. Helix Is Right-Handed
  • 34.
  • 35. Biologically Significant Form = B-DNA
    Low Salt = Hydrated, 10.5 bp/turn
  • 36. Side-view
    Top-view
    A- DNA Exists Under High Salt Conditions
    Base pairs tilted, 23 Å, 11bp/turn
  • 37. Z-DNA Is a Left-Handed Helix
    Zig-zag conformation, 18 Å, 12 bp/turn,
    no major groove
  • 38.
  • 39. Propeller Twist Results from Bond Rotation
  • 40. Sugar Conformations
    Ideal B-DNA is C2'-endo (South) Ideal A-RNA is C3'-endo (North)
  • 41. anti and syn conformational ranges for glycosydic bonds in pyrimidine (left) and purine (right) nucleosides
    Source: Blackburn and Gait, Nucleic acids in chemistry and biology, Oxford University Press New York 1996.
  • 42. Syn vs. Anti Conformations
    Syn conformation causes left-handed helix
  • 43. Syn-Anti Bond Rotation
  • 44.
  • 45. Reassociation Kinetics
  • 46. Denaturation of DNA Strands and the Hyperchromic Shift
    Denaturation (melting) is the breaking of H, but not covalent, bonds in DNA double helix  duplex unwinds  strands separate
    Viscosity decreases and bouyant density increases
    Hyperchromic shift – uv absorption increases with denaturation of duplex
    Basis for melting curves because G-C pairs have three H bonds but A-T pairs have only two H bonds
    Duplexes with high G-C content have a higher melting temperature because G-C pairs require a higher temperature for denaturation
  • 47.
  • 48. Molecular Hybridization
    Reassociation of denatured strands
    Occurs because of complementary base pairing
    Can form RNA-DNA Hybrids
    Can detect sequence homology between species
    Basis for in situ hybridization, Southern and Northern blotting, and PCR
  • 49. Hybridization
  • 50. Reassociation Kinetics
    Derive information about the complexity of a genome
    To study reassociation, genome must first be fragmented (e.g. by shear forces)
    Next, DNA is heat-denatured
    Finally, temperature is slowly lowered and rate of strand reassociation (hybridization) is monitored
  • 51. Data Analysis
    Pieces of DNA collide randomly and hybridize if complementary
    Plot the % reassociation versus the log of the product of the concentration of single-stranded (ss) DNA and time
    Reassociation follows second order kinetics: C/C0 = 1/1 + k C0t
    Initially, C = C0 that is [ssDNA] = 100%
    As time elapses, C approaches a [ssDNA] of 0%
  • 52. Initially there is a mixture of unique DNA sequence fragments so hybridization occurs slowly. As this pool shrinks, hybridization occurs more quickly
    C0t1/2= half-reaction time or the point where one half of the DNA is present as ds fragments and half is present as ss fragments
    If all pairs of ssDNA hybrids contain unique sequences and all are about the same size, C0t1/2is directly proportional to the complexity of the DNA
    Complexity = X represents the length in nucleotide pairs of all unique DNA fragments laid end to end
    Assuming that the DNA represents the entire genome and all sequences are different from each other, then X = the size of the haploid genome
  • 53. The Tm
  • 54. Maximum denaturation =
    100% single stranded
    50% double,
    50% single
    stranded
    Double stranded
    The Hyperchromic Shift (Melting Curve Profile)
    Tm = temperature at which 50% of DNA is denatured
  • 55. High G-C Content Results in a Genome of Greater Bouyant Density
  • 56. 100% ssDNA
    100% dsDNA
    Ideal C0t Curve
  • 57. Largest genome
    Smallest
    genome
    Larger genomes take longer to reassociate because there are more DNA fragments to hybridize
  • 58. C0t1/2 Is Directly Proportional to Genome Size
  • 59. 0
    Highly repetitive DNA
    Moderately
    repetitive DNA
    Fraction remaining
    single-stranded (C/C0)
    Unique DNA sequences
    100
    10-4
    10-2
    100
    102
    104
    C0t (moles x sec/L)
    Genomes are composed of unique, moderately repetitive and highly repetitive sequences
  • 60. More complex genomes contain more classes of DNA sequences
  • 61. G-C Content Increases Tm
  • 62. DNA Topology
    Some of the following slides and text are taken from the DNA Topology lecture from Doug Brutlag’s January 7, 2000 Biochemistry 201 Advanced Molecular Biology Course at Stanford University
  • 63. What Is Supercoiling & Why Should I Care?
    DNA forms supercoils in vivo
    Important during replication and transcription
    Topology only defined for a continuous strand - no strand breakage
    Numerical expression for degree of supercoiling:
    Lk = Tw + Wr
    L:linking number, # of times that one DNA strand winds about the others strands - is always an integer
    T: twist, # of revolutions about the duplex helix
    W: writhe, # of turns of the duplex axis about the superhelical axis is by definition the measure of the degree of supercoiling
  • 64. DNA Topology
    Supercoiling or writhing of circular DNA is a result of the DNA being underwoundwith respect to the relaxed form of DNA
    There are actually fewer turns in the DNA helix than would be expected given the natural pitch of DNA in solution (10.4 base pairs per turn)
    When a linear DNA is free in solution it assumes a pitch which contains 10.4 base pairs per turn
    This is less tightly wound than the 10.0 base pairs per turn in the Watson and Crick B-form DNA
  • 65. DNA that is underwound is referred to as negatively supercoiled
    The helices wind about each other in a right-handed path in space
    DNA that is overwound will relax and become a positively supercoiled DNA helix
    Positively coiled DNA has its DNA helices wound around each other in a left-handed path in space
  • 66. DNA topology
  • 67. Linking number - # times would have to pass cccDNA strand through the other to entirely separate the strands and not break any covalent bonds
    Twist - # times one strand completely wraps (# helical turns) around the other strand
    Writhe – when long axis of double helix crosses over itself (causes torsional stress)
  • 68. Linking Defined
    Linking number, Lk, is the total number of times one strand of the DNA helix is linked with the other in a covalently closed circular molecule
  • 69. The linking number is only defined for covalently closed DNA and its value is fixed as long as the molecule remains covalently closed.
    The linking number does not change whether the covalently closed circle is forced to lie in a plane in a stressed conformation or whether it is allowed to supercoil about itself freely in space.
    The linking number of a circular DNA can only be changed by breaking a phosphodiester bond in one of the two strands, allowing the intact strand to pass through the broken strand and then rejoining the broken strand.
    Lkis always an integer since two strands must always be wound about each other an integral number of times upon closure.
  • 70. Linking Number, Twistsand Writhe
  • 71. DNA tied up in knots
    Metabolic events involving unwinding impose great stress on the DNA because of the constraints inherent in the double helix
    There is an absolute requirement for the correct topological tension in the DNA (super-helical density) in order for genes to be regulated and expressed normally
    For example, DNA must be unwound for replication and transcription
    Figure from Rasika Harshey’s lab at UT Austin showing an enhancer protein (red) bound to the DNA in a specific interwrapped topology that is called a transposition synapse.www.icmb.utexas.edu/.../47_Topology_summary.jpg
  • 72. Knots, Twists, Writhe and Supercoiling
    Circular DNA chromosomes, from viruses for instance, exist in a highly compact or folded conformation
  • 73. Twist
    The linking number of a covalently closed circular DNA can be resolved into two components called the twists, Tw and the writhes, Wr.
    Lk = Tw + Wr
    The twists are the number of times that the two strands are twisted about each other
    The length and pitch of DNA in solution determine the twist. [Tw = Length (bp)/Pitch (bp/turn)]
  • 74. Writhe
    Writhe is the number of times that the DNA helix is coiled about itself in three-dimensional space
    The twist and the linking number, determine the value of the writhe that forces the DNA to assume a contorted path is space. [Wr = Lk - Tw ]
  • 75. Unlike the Twist and the Linking number, the writhe of DNA only depends on the path the helix axis takes in space, not on the fact that the DNA has two strands
    If the path of the DNA is in a plane, the Wr is always zero
    If the path of the DNA helix were on the surface of a sphere (like the seams of a tennis ball or base ball) then the total Writhe can also be shown to be zero
  • 76. Molecules that differ by one unit in linking number can be separated by electrophoresis in agarose due to the difference in their writhe (that is due to difference in folding).
    The variation in linking number is reflected in a difference in the writhe.
    The variation in writhe is subsequently reflected in the state of compaction of the DNA molecule.
  • 77. Interwound
    Toroidal
    Writhe of supercoiled DNA
  • 78. Types of Supercoils
  • 79. Supercoiling
  • 80. Negative vs. Positive Supercoiling
    Right handed supercoiling = negative supercoiling (underwinding)
    Left handed supercoiling = positive supercoiling
    Relaxed state is with no bends
    DNA must be constrained: plasmid DNA or by proteins
    Unraveling the DNA at one position changes the superhelicity
  • 81. Relaxed
    Supertwisted
  • 82. Unwinding DNA
  • 83. Toposomerase
  • 84. Topoisomerase II makes ds breaks
  • 85. Topoisomerase I makes ss breaks
  • 86.
  • 87.
  • 88.
  • 89.
  • 90.
  • 91.
  • 92. Ability of Uracil To Form Stable Base Pairs Enhances RNA’s Ability To Form Stem-loop Structures
  • 93. Intercalating Agents: Ethidium Bromide
    By electrophoresing supercoiled DNA in the presence of an intercalating agent such as ethidium bromide, one can distinguish negatively supercoiled DNA from positively supercoiled DNA
    When negatively supercoiled DNA binds an intercalating agent, the average pitch is reduced because the twist angle between adjacent base pairs on either side of the intercalating agent is reduced
    Reduction of twist causes a compensatory increase in writhe in a covalently closed molecule. Thus, a molecule that is initially negatively supercoiled will become more relaxed and a positively supercoiled molecule will become more twisted.
  • 94. Histone Variants
    Alter nucleosome function
    H2A.z often found in areas with transcribed regions of DNA
    prevents nucleosome from forming repressive structures that would inhibit access of RNA polymerase
    Mark areas of chromatin with alternate functions
    CENP-A replaces H3
    Associated with nucleosomes that contain centromeric DNA
    Has longer N-terminal tail that may function to increase binding sites available for kinetochore protein binding
  • 95. more peripheral
    more central
    Unwrapping of DNA from nucleosome allows DNA-binding proteins access to their binding sites
    Many DNA-binding proteins require histone-free DNA
    DNA-histone interactions dynamic: unwrapping is spontaneous and intermittent
    Accessibility to binding protein sites dependent on location in nucleosomal DNA
    more central sites less accessible than those near the ends decreasing probability of protein binding and hence regulating transcriptional activity
  • 96. Nucleosome remodeling complexes
    Alter stability of DNA-histone interaction to increase accessibility of DNA
    Change nucleosome location
    Require ATP
    3 mechanisms:
    Slide histone octamer along DNA
    Transfer histone octamer to another DNA
    Remodel to increase access to DNA
  • 97. DNA-binding protein dependent nucleosome positioning
    Nucleosomes are sometimes specifically positioned
    Keeps DNA-binding protein site in linker region (hence accessible)
    Can be directed by DNA-binding proteins or by specific sequences
    Usually involves competition between nucleosomes and binding proteins
    If proteins are positioned such that less than 147 bp exists between them, nucleosomes cannot associate
  • 98. Positioning can be inhibitory
    Some proteins can bind to DNA and a nucleosome
    By putting a tightly bound binding protein next to a nucleosome, additional nucleosomes will assemble immediately adjacent to the protein preferentially
  • 99. DNA sequences can direct positioning
    DNA sequences that position nucleosomes are A-T or G-C rich because DNA is bent in nucleosomes
    By alternating A-T or G-C rich sequences, can change the position in which the minor groove faces the histone octamer
    These sequences are rare
  • 100. Majority of nucleosomes are not positioned
    Tightly positioned nucleosomes are usually associated with areas for transcription initiation
    Positioned nucleosomes can prevent or enhance access to DNA sequences needed for binding protein attachment
  • 101. Modification of N-terminal tails
    Results in increased or decreased affinity of nucleosome for DNA
    Modifications include acetylation, methylation and phosphorylation
    Combination of modifications may encode information for gene expression (positively or negatively
  • 102. Acetylated nucleosomes are associated with actively transcribed areas because reduces the affinity of the nucleosome for DNA
    Deacetylation associated with inactive transcription units
    Phosphorylation also increases transcription
    Like acetylation, phosphorylation reduces positive charge on histone proteins
    Methylation represses transcription
    Also affects ability of nucleosome array to form higher order structures
  • 103.
  • 104. HAT
    Acetylation creates binding
    sites for bromo- and chromodomain
    protein binding
  • 105. Chromatin remodeling complexes and histone modifying enzymes work together to make DNA more accessible
  • 106. Distributive inheritance of old histones
    Old histones have to be inherited to maintain histone modifications and appropriate gene expression
    H3▪H4 tetramers are randomly transferred to new daughter strand, never put into soluble pool
    H2A▪H2B dimers are put into pool and compete for association with H3▪H4 tetramers
  • 107.
  • 108. Histone assembly requires chaperones
    Assembly of nucleosome is not spontaneous
    Chaperone proteins are needed to bring in free dimers and tetramers after replication fork has been passed
    Chaperones are associated with PCNA, the sliding clamp protein of eukaryotic replication, immediately after PCNA is released by DNA polymerase
  • 109. Nucleotides and primer:template junction are essential substrates for DNA synthesis

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