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. Structure and Analysis of DNA ©2000 Timothy G. Standish Introduction The Central Dogma of Molecular Biology Cell DNA Transcription Translation mRNA Ribosome Polypeptide (protein) ©1998 Timothy G. Standish Outline 1 How we know DNA is the genetic material 2 Basic structure of DNA and RNA 3 Ways in which DNA can be studied and what they tell us about genomes ©2000 Timothy G. Standish Historical Events • • • • • • 1869 Friedrich Miescher identified DNA, which he called nuclein, from pus cells 1889 Richard Altman renamed nuclein nucleic acid 1928 Griffith discovered that genetic information could be passed from one bacteria to another; known as the transforming principle 1944 Avery showed that the transforming material was pure DNA not protein, lipid or carbohydrate. 1952 Hershey and Chase used bacteriophage (virus) and E. coli to show that only viral DNA entered the host 1953 Watson and Crick discovered the structure of DNA was a double helix ©2000 Timothy G. Standish Transformation Of Bacteria Two Strains Of Streptococcus Rough Strain (Harmless) Capsules Smooth Strain (Virulent) ©2000 Timothy G. Standish Transformation Of Bacteria The Griffith’s 1928 Experiment OUCH! + Control - Control - Control Experimental ©2000 Timothy G. Standish Avery, MacLeod and McCarty 1944 Avery, MacLeod and McCarty repeated Griffith’s 1928 experiment with modifications designed to discover the “transforming factor” After extraction with organic solvents to eliminate lipids, remaining extract from heat killed cells was digested with hydrolytic enzymes specific for different classes of macro molecules: Enzyme Transformation? Protease Yes Saccharase Yes Nuclease No ©2000 Timothy G. Standish The Hershey-Chase Experiement The Hershey-Chase experiment showed definitively that DNA is the genetic material Hershey and Chase took advantage of the fact that T2 phage is made of only two classes of macromolecules: Protein and DNA H H2N C C CH2 CH2 S CH3 H O H2N C C OH Methionine CH2 SH OH O OH Cysteine Some amino acids contain sulfur, thus proteins contain sulfur, but not phosphorous. HO P NH2 O O OH H Nucleotides contain phosphorous, thus DNA contains phosphorous, but not sulfur. ©2000 Timothy G. Standish S35 T2 grown in containing media incorporate S35 into their proteins Using S35 Bacteria grown in T2 attach to bacteria and inject genetic material normal nonradioactive media When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet The supernatant is radioactive, but the pellet is not. Did protein enter the bacteria? Blending causes phage protein coat to fall off Is protein the genetic material? P32 T2 grown in containing media incorporate P32 into their DNA Using P32 Bacteria grown in T2 attach to bacteria and inject genetic material normal nonradioactive media When centrifuged, phage protein coats remain in the supernatant while bacteria form a pellet The pellet is radioactive, but the supernatant is not. Did DNA enter the bacteria? Blending causes phage protein coat to fall off Is DNA the genetic material? A Nucleotide Adenosine Mono Phosphate (AMP) Phosphate HO H+ Nucleotide OH P O Base N H O 5’CH2 4’ NH2 H N O 1’ Sugar 3’ OH 2’ H OH N N Nucleoside Purines NH2 Adenine N N N O CH3 (DNA) N Guanine NH N Thymine O NH2 Uracil (RNA) NH N O N N Pyrimidines NH O N O NH2 Cytosine N N O Base Pairing Guanine And Cytosine - + + + - Base Pairing Adenine And Thymine + - Adenine - + Thymine Base Pairing Adenine And Cytosine + - - Base Pairing Guanine And Thymine + + Some minor purine and pyrimidine bases ©2000 Timothy G. Standish 5’Phosphate group P HO NH2 O N O CH2 OH N N O H N O CH2 O HO P O O N O CH2 OH H H2O NH N O HO P O H O NH2 N O CH2 O H O H N O O CH2 O P HO H O OH 5’Phosphate group HO CH2 3’Hydroxyl group H2O N O O P NH2 HO P O H O HO O D N A 3’Hydroxyl group OH - - - - - - G - 3.4 nm 1 nm - - Minor groove C G C T A A T - The Watson - Crick Model Of DNA G C T A C G A T Major groove A T C G G C 0.34 nm T A - - - - - - - - - - - ©2000 Timothy G. Standish - B DNA Forms of the Double Helix G T A Minor groove Z DNA A T C G C A DNA 3.9 nm 1 nm Minor groove G C T A C G A T Major groove A T G T A 1.2 nm 2.8 nm 0.9 nm 6.8 nm Major groove 0.57 nm C G C 0.26 nm 0.34 nm 10.4 Bp/turn +34.6o Rotation/Bp 11 Bp/turn +34.7o Rotation/Bp 12 Bp/turn -30.0o Rotation/Bp ©2000 Timothy G. Standish . ©2000 Timothy G. Standish . A-DNA:1. Large hole in center 2. Sugar phosphate backbone is at the edge 3. Bases are displaced towards edge B-DNA-1. Bases in center (no hole) 2. Phosphates at periphery Z-DNA-1. Bases present throughout the matrix of the helix 2. No exclusive domains for either bases or backbone 3. Left hand helix ©2000 Timothy G. Standish Biological Significance A-DNA-occurs only in dehydrated samples of DNA, such as those used in crystallographic experiments, and possibly is also assumed by DNA-RNA hybrid helices and by regions of double-stranded RNA. Z-DNA has been found, it is commonly believed to provide torsional strain relief (supercoiling) while DNA transcription occurs. The potential to form a Z-DNA structure also correlates with regions of active transcription ©2000 Timothy G. Standish Even More Forms Of DNA C-DNA: – Exists only under high dehydration conditions – 9.3 bp/turn, 0.19 nm diameter and tilted bases B-DNA appears to be the – Occurs in helices lacking guanine most common form in – 8 bp/turn vivo. However, under some circumstances, E-DNA: – Like D-DNA lack guanine alternative forms of DNA – 7.5 bp/turn may play a biologically P-DNA: significant role. D-DNA: – Artificially stretched DNA with phosphate groups found inside the long thin molecule and bases closer to the outside surface of the helix – 2.62 bp/turn ©2000 Timothy G. Standish Certain DNA sequences adopt unusual structures Palindrome: The term is applied to regions of DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA. Such sequences are self-complementary within each strand and therefore have the potential to form hairpin or cruciform (cross-shaped) structures ©2000 Timothy G. Standish Certain DNA sequences adopt unusual structures Mirror repeats :When the inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat. Mirror repeats do not have complementary sequences within the same strand and cannot form hairpin or cruciform structures. ©2000 Timothy G. Standish Certain DNA sequences adopt unusual structures ©2000 Timothy G. Standish . ©2000 Timothy G. Standish . ©2000 Timothy G. Standish keto-enol tautomerism keto-enol tautomerism refers to a chemical equlibrium between a keto form (a ketone or an aldehyde) and an enol (An alcohol) In DNA, the nucleotide bases are in keto form. Rare enol tautomers of the bases G and T can lead to mutation because of their altered base-pairing properties. ©2000 Timothy G. Standish Triplex DNA Nucleotides participating in a WatsonCrick base pair can form a number of additional hydrogen bonds, particularly with functional groups arrayed in the major groove. For example, a cytidine residue (if protonated) can pair with the guanosine residue of a G-C nucleotide pair. ©2000 Timothy G. Standish Triplex DNA The N-7, O6, and N6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA, are often referred to as Hoogsteen positions, and the non-Watson-Crick pairing is called Hoogsteen pairing. The triplexes form most readily within long sequences containing only pyrimidines or only purines in a given strand Four DNA strands can also pair to form a tetraplex ©2000 Timothy G. Standish . ©2000 Timothy G. Standish H-DNA A particularly exotic DNA structure, known as H-DNA, is found in polypyrimidine or polypurine tracts that also incorporate a mirror repeat. A simple example is a long stretch of alternating T and C residues ©2000 Timothy G. Standish H-DNA ©2000 Timothy G. Standish Structure of RNA The single strand of RNA tends to assume a right-handed helical conformation dominated by base stacking Interactions ,which are strongest between two purines The purine-purine interaction is so strong that a pyrimidine separating two purines is often displaced from the stacking pattern so that the purines can interact ©2000 Timothy G. Standish Structure of RNA RNA can base-pair with complementary regions of either RNA or DNA. For DNA: G pairs with C and A pairs with U ,however base pairing between G and U is fairly common in RNA. Where complementary sequences are present, the predominant double-stranded structure is an A-form right-handed double helix. Hairpin loops form between nearby selfcomplementary sequences. ©2000 Timothy G. Standish . short base sequences (such as UUCG) are often found at the ends of RNA hairpins and are known to form particularly tight and stable loops. Additional structural contributions are made by hydrogen bonds that are not part of standard Watson-Crick base pairs. For example, the 2-hydroxyl group of ribose can hydrogen-bond with other groups. rRNA has a characteristic secondary structure due to many intramolecular Hbonds ©2000 Timothy G. Standish Structure Of t-RNA ©2000 Timothy G. Standish Denaturation and Renaturation Heating double stranded DNA can overcome the hydrogen bonds holding it together and cause the strands to separate resulting in denaturation of the DNA When cooled relatively weak hydrogen bonds between bases can reform and the DNA renatures Denatured DNA ATGAGCTGTACGATCGTG ATGAGCTGTACGATCGTG TACTCGACATGCTAGCAC Double stranded DNA ATGAGCTGTACGATCGTG TACTCGACATGCTAGCAC TACTCGACATGCTAGCAC Single stranded DNA Double stranded DNA ©2000 Timothy G. Standish Denaturation and Renaturation DNA with a high guanine and cytosine content has relatively more hydrogen bonds between strands This is because for every GC base pair 3 hydrogen bonds are made while for AT base pairs only 2 bonds are made Thus higher GC content is reflected in higher melting or denaturation temperature ACGAGCTGCACGAGC TGCTCGACGTGCTCG ATGATCTGTAAGATC TACTAGACATTCTAG 67 % GC content High melting temperature 33 % GC content Low melting temperature ATGAGCTGTCCGATC TACTCGACAGGCTAG 50 % GC content - Intermediate melting temperature ©2000 Timothy G. Standish Determination of GC Content Comparison of melting temperatures can be used to determine the GC content of an organisms genome To do this it is necessary to be able to detect whether DNA is melted or not Absorbance at 260 nm of DNA in solution provides a means of determining how much is single stranded Single stranded DNA absorbs 260 nm ultraviolet light more strongly than double stranded DNA does although both absorb at this wavelength Thus, increasing absorbance at 260 nm during heating indicates increasing concentration of single stranded DNA ©2000 Timothy G. Standish Determination of GC Content 1.0 Tm is the temperature at which half the DNA is melted OD260 Single stranded DNA Relatively low GC content Relatively high GC content Tm = 75 oC Tm = 85 oC Double stranded DNA 0 65 70 75 80 85 Temperature (oC) 90 95 ©2000 Timothy G. Standish GC Content Of Some Genomes Organism % GC Homo sapiens 39.7 % Sheep 42.4 % Hen 42.0 % Turtle 43.3 % Salmon 41.2 % Sea urchin 35.0 % E. coli 51.7 % Staphylococcus aureus 50.0 % Phage l Phage T7 55.8 % 48.0 % ©2000 Timothy G. Standish Hybridization The bases in DNA will only pair in very specific ways, G with C and A with T In short DNA sequences, imprecise base pairing will not be tolerated Long sequences can tolerate some mispairing only if -G of the majority of bases in a sequence exceeds the energy required to keep mispaired bases together Because the source of any single strand of DNA is irrelevant, merely the sequence is important, DNA from different sources can form double helix as long as their sequences are compatible Thus, this phenomenon of base pairing of single stranded DNA strands to form a double helix is called hybridization as it may be used to make hybrid DNA composed of strands which came from different sources ©2000 Timothy G. Standish Hybridization DNA from source “X” CTGATGGTCATGAGCTGTCCGATCGATCAT TACTCGACAGGCTAG Hybridization TACTCGACAGGCTAG DNA from source “Y” ©2000 Timothy G. Standish Hybridization Because DNA sequences will seek out and hybridize with other sequences with which they base pair in a specific way much information can be gained about unknown DNA using single stranded DNA of known sequence Short sequences of single stranded DNA can be used as “probes” to detect the presence of their complimentary sequence in any number of applications including: – Southern blots – Northern blots (in which RNA is probed) – In situ hybridization – Dot blots . . . In addition, the renaturation or hybridization of DNA in solution can tell much about the nature of organism’s genomes ©2000 Timothy G. Standish Reassociation Kinetics An organism’s DNA can be heated in solution until it melts, then cooled to allow DNA strands to reassociate forming double stranded DNA This is typically done after shearing the DNA to form many fragments a few hundred bases in length. The larger and more complex an organisms genome is, the longer it will take for complimentary strands to bum into one another and hybridize Rate of reassociation is proportional to concentration of the two homologus dissociated strands. Reassociation follows second order kinetics: , now integrate this equation dt/dc = -kc2 ©2000 Timothy G. Standish Reassociation Kinetics The following equation describes the second order rate kinetics of DNA reassociation: Concentration of single stranded DNA after time t Initial concentration of single stranded DNA C 1 = Co 1 + kCot Second order rate constant (the important thing is that it is a constant) Co (measured in moles/liter) x t (seconds). Generally graphed on a log10 scale. Cot1/2 is the point at which half the initial concentration of single stranded DNA has annealed to form double-stranded DNA ©2000 Timothy G. Standish Cot 0.5 value Cot0.5 value is proportional to complexity of the genome. A plot of C/Co against Cot is called Cot curve and it provides information about complexity of a genome. ©2000 Timothy G. Standish Genome complexity Complexity is the minimum length of DNA that contains a single copy of all the single reiterated sequences that are represented within the genome. Complexity of a genome is equal to its molecular mass only if a genome has unique nucleotide sequences (repetitive sequences absent). ©2000 Timothy G. Standish example # For a hypothetical DNA-1 having three nucleotide sequences, N1, N2, N3. Molecular mass=N1+N2+N3 Complixity=N1+N2+N3 # For a hypothetical DNA-2 having 103 copies of N1 ,105 copies of N2 & 1 copy of N3. Molecular mass= 103 N1+ 105 N2 + N3 Complixity=N1+N2+N3 ©2000 Timothy G. Standish Reassociation Kinetics 1.0 Fraction remaining singlestranded (C/Co) 0.5 0 Higher Cot1/2 values indicate greater genome complexity Cot1/2 10-4 10-3 10-2 10-1 1 101 102 103 Cot (mole x sec./l) 104 ©2000 Timothy G. Standish Reassociation Kinetics 1.0 Prokaryotic DNA Fraction remaining Repetitive singleDNA stranded (C/Co) 0.5 Unique sequence complex DNA Eukaryotic DNA 0 10-4 10-3 10-2 10-1 1 101 102 103 Cot (mole x sec./l) 104 ©2000 Timothy G. Standish Repetitive DNA Organism % Repetitive DNA Homo sapiens 21 % Mouse 35 % Calf 42 % Drosophila 70 % Wheat 42 % Pea 52 % Maize 60 % Saccharomycetes cerevisiae 5% E. coli 0.3 % ©2000 Timothy G. Standish ©2000 Timothy G. Standish