Dna lecture

Nitrogenous Bases and Pentose Sugars

Purine and Pyrimidine Structure(1) Pyrimidines are planar (2) Purines are nearly planar(3) Numbering is different

Bases Have Tautomeric FormsUracil

Glycosidic bondNucleosides vs. Nucleotides

Nucleotides formed by condensation reactions

Only RNA Is Hydrolyzed by Base

Nucleoside Diphosphate and Triphosphate

Ester bondsDinucleotides and Polynucleotides

Other Base Pairs Are PossibleWatson-Crick, Reverse Watson-Crick, Hoogsteen, Reverse Hoogsteen, Wobble, Reverse WobbleHomo PurinesHetero Purines

Base Pairing Can Result in Alternative DNA StructuresTriplexTetraplexCruciformHairpin Loop

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.

DNase can only cleave external bond demonstrating periodicity

Watson and Crick Model (1953)HydrophilicHydrophobicComplementarity2 long polynucleotide chains coiled around a central axisBases are 3.4 Å (0.34 nm) apart on inside of helixBases flat & lie perpendicular to the axisComplete turn = 34 Å10 bases/turnDiameter = 20 ÅAlternating major and minor grooves

Base Pairing Results from H-BondsOnly A=T and GC yield 20 Å Diameter

…Because of Base Pair Torsional Bond Angles

Base Pairs and Groove Formation

Biologically Significant Form = B-DNALow Salt = Hydrated, 10.5 bp/turn

Side-viewTop-viewA- DNA Exists Under High Salt ConditionsBase pairs tilted, 23 Å, 11bp/turn

Z-DNA Is a Left-Handed HelixZig-zag conformation, 18 Å, 12 bp/turn, no major groove

Propeller Twist Results from Bond Rotation

Sugar ConformationsIdeal B-DNA is C2'-endo (South) Ideal A-RNA is C3'-endo (North)

anti and syn conformational ranges for glycosydic bonds in pyrimidine (left) and purine (right) nucleosidesSource: Blackburn and Gait, Nucleic acids in chemistry and biology, Oxford University Press New York 1996.

Syn vs. Anti ConformationsSyn conformation causes left-handed helix

Denaturation of DNA Strands and the Hyperchromic ShiftDenaturation (melting) is the breaking of H, but not covalent, bonds in DNA double helix  duplex unwinds  strands separateViscosity decreases and bouyant density increasesHyperchromic shift – uv absorption increases with denaturation of duplexBasis for melting curves because G-C pairs have three H bonds but A-T pairs have only two H bondsDuplexes with high G-C content have a higher melting temperature because G-C pairs require a higher temperature for denaturation

Molecular HybridizationReassociation of denatured strandsOccurs because of complementary base pairing Can form RNA-DNA HybridsCan detect sequence homology between speciesBasis for in situ hybridization, Southern and Northern blotting, and PCR

Reassociation KineticsDerive information about the complexity of a genomeTo study reassociation, genome must first be fragmented (e.g. by shear forces)Next, DNA is heat-denaturedFinally, temperature is slowly lowered and rate of strand reassociation (hybridization) is monitored

Data AnalysisPieces of DNA collide randomly and hybridize if complementaryPlot the % reassociation versus the log of the product of the concentration of single-stranded (ss) DNA and timeReassociation follows second order kinetics: C/C0 = 1/1 + k C0tInitially, C = C0 that is [ssDNA] = 100%As time elapses, C approaches a [ssDNA] of 0%

Initially there is a mixture of unique DNA sequence fragments so hybridization occurs slowly. As this pool shrinks, hybridization occurs more quicklyC0t1/2= half-reaction time or the point where one half of the DNA is present as ds fragments and half is present as ss fragmentsIf all pairs of ssDNA hybrids contain unique sequences and all are about the same size, C0t1/2is directly proportional to the complexity of the DNAComplexity = X represents the length in nucleotide pairs of all unique DNA fragments laid end to endAssuming that the DNA represents the entire genome and all sequences are different from each other, then X = the size of the haploid genome

Maximum denaturation = 100% single stranded50% double, 50% single strandedDouble strandedThe Hyperchromic Shift (Melting Curve Profile)Tm = temperature at which 50% of DNA is denatured

High G-C Content Results in a Genome of Greater Bouyant Density

100% ssDNA100% dsDNAIdeal C0t Curve

Largest genomeSmallest genomeLarger genomes take longer to reassociate because there are more DNA fragments to hybridize

C0t1/2 Is Directly Proportional to Genome Size

0Highly repetitive DNAModerately repetitive DNAFraction remainingsingle-stranded (C/C0)Unique DNA sequences10010-410-2100102104C0t (moles x sec/L)Genomes are composed of unique, moderately repetitive and highly repetitive sequences

More complex genomes contain more classes of DNA sequences

DNA TopologySome 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

What Is Supercoiling & Why Should I Care?DNA forms supercoils in vivoImportant during replication and transcriptionTopology only defined for a continuous strand - no strand breakageNumerical 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

DNA TopologySupercoiling or writhing of circular DNA is a result of the DNA being underwoundwith respect to the relaxed form of DNAThere 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

DNA that is underwound is referred to as negatively supercoiledThe helices wind about each other in a right-handed path in spaceDNA that is overwound will relax and become a positively supercoiled DNA helixPositively coiled DNA has its DNA helices wound around each other in a left-handed path in space

Linking number - # times would have to pass cccDNA strand through the other to entirely separate the strands and not break any covalent bondsTwist - # times one strand completely wraps (# helical turns) around the other strandWrithe – when long axis of double helix crosses over itself (causes torsional stress)

Linking DefinedLinking 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

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.

Linking Number, Twistsand Writhe

DNA tied up in knotsMetabolic events involving unwinding impose great stress on the DNA because of the constraints inherent in the double helixThere is an absolute requirement for the correct topological tension in the DNA (super-helical density) in order for genes to be regulated and expressed normallyFor example, DNA must be unwound for replication and transcriptionFigure 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

Knots, Twists, Writhe and SupercoilingCircular DNA chromosomes, from viruses for instance, exist in a highly compact or folded conformation

TwistThe linking number of a covalently closed circular DNA can be resolved into two components called the twists, Tw and the writhes, Wr.Lk = Tw + WrThe twists are the number of times that the two strands are twisted about each otherThe length and pitch of DNA in solution determine the twist. [Tw = Length (bp)/Pitch (bp/turn)]

WritheWrithe is the number of times that the DNA helix is coiled about itself in three-dimensional spaceThe 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 ]

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

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.

InterwoundToroidalWrithe of supercoiled DNA

Negative vs. Positive SupercoilingRight handed supercoiling = negative supercoiling (underwinding)Left handed supercoiling = positive supercoilingRelaxed state is with no bends DNA must be constrained: plasmid DNA or by proteinsUnraveling the DNA at one position changes the superhelicity

Topoisomerase II makes ds breaks

Topoisomerase I makes ss breaks

Ability of Uracil To Form Stable Base Pairs Enhances RNA’s Ability To Form Stem-loop Structures

Intercalating Agents: Ethidium BromideBy electrophoresing supercoiled DNA in the presence of an intercalating agent such as ethidium bromide, one can distinguish negatively supercoiled DNA from positively supercoiled DNAWhen 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 reducedReduction 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.

Histone VariantsAlter nucleosome functionH2A.z often found in areas with transcribed regions of DNA prevents nucleosome from forming repressive structures that would inhibit access of RNA polymeraseMark areas of chromatin with alternate functionsCENP-A replaces H3Associated with nucleosomes that contain centromeric DNAHas longer N-terminal tail that may function to increase binding sites available for kinetochore protein binding

more peripheralmore centralUnwrapping of DNA from nucleosome allows DNA-binding proteins access to their binding sitesMany DNA-binding proteins require histone-free DNADNA-histone interactions dynamic: unwrapping is spontaneous and intermittentAccessibility to binding protein sites dependent on location in nucleosomal DNAmore central sites less accessible than those near the ends decreasing probability of protein binding and hence regulating transcriptional activity

Nucleosome remodeling complexesAlter stability of DNA-histone interaction to increase accessibility of DNAChange nucleosome locationRequire ATP3 mechanisms:Slide histone octamer along DNATransfer histone octamer to another DNARemodel to increase access to DNA

DNA-binding protein dependent nucleosome positioningNucleosomes are sometimes specifically positionedKeeps DNA-binding protein site in linker region (hence accessible)Can be directed by DNA-binding proteins or by specific sequencesUsually involves competition between nucleosomes and binding proteinsIf proteins are positioned such that less than 147 bp exists between them, nucleosomes cannot associate

Positioning can be inhibitorySome proteins can bind to DNA and a nucleosomeBy putting a tightly bound binding protein next to a nucleosome, additional nucleosomes will assemble immediately adjacent to the protein preferentially

DNA sequences can direct positioningDNA sequences that position nucleosomes are A-T or G-C rich because DNA is bent in nucleosomesBy alternating A-T or G-C rich sequences, can change the position in which the minor groove faces the histone octamerThese sequences are rare

Majority of nucleosomes are not positionedTightly positioned nucleosomes are usually associated with areas for transcription initiationPositioned nucleosomes can prevent or enhance access to DNA sequences needed for binding protein attachment

Modification of N-terminal tailsResults in increased or decreased affinity of nucleosome for DNAModifications include acetylation, methylation and phosphorylationCombination of modifications may encode information for gene expression (positively or negatively

Acetylated nucleosomes are associated with actively transcribed areas because reduces the affinity of the nucleosome for DNADeacetylation associated with inactive transcription unitsPhosphorylation also increases transcriptionLike acetylation, phosphorylation reduces positive charge on histone proteinsMethylation represses transcriptionAlso affects ability of nucleosome array to form higher order structures

HATAcetylation creates binding sites for bromo- and chromodomain protein binding

Chromatin remodeling complexes and histone modifying enzymes work together to make DNA more accessible

Distributive inheritance of old histonesOld histones have to be inherited to maintain histone modifications and appropriate gene expressionH3▪H4 tetramers are randomly transferred to new daughter strand, never put into soluble poolH2A▪H2B dimers are put into pool and compete for association with H3▪H4 tetramers

Histone assembly requires chaperonesAssembly of nucleosome is not spontaneousChaperone proteins are needed to bring in free dimers and tetramers after replication fork has been passedChaperones are associated with PCNA, the sliding clamp protein of eukaryotic replication, immediately after PCNA is released by DNA polymerase

Nucleotides and primer:template junction are essential substrates for DNA synthesis

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