DNA Structure I & II PDF

HUBI 2004: Fundamentals of Modern Molecular Biology DNA structure I & II Pavan K. Kakumani January 09 and 13, 2025 1  Learning Objectives What is the composition of a DNA nucleo...

HUBI 2004: Fundamentals of Modern Molecular Biology DNA structure I & II Pavan K. Kakumani January 09 and 13, 2025 1  Learning Objectives What is the composition of a DNA nucleotide? What are the types of DNA bases? The two rules followed by a DNA double helix, to form a stable structure. Salient features of the 3D structure of B-DNA. What are major and minor grooves of a DNA double helix? What is DNA supercoiling and how it is regulated by enzymes called topoisomerases? Explain the differences between the major structural forms of DNA.  Molecular Biology Jim Watson Francis Crick 1953 - structure of DNA The Nobel Prize in Physiology or Medicine in 1962 was awarded to James Watson, Francis Crick and Maurice Wilkins for their discovery of the molecular structure of DNA, which helped solve one of the most important of all biological riddles. https://www.nobelprize.org/prizes/medicine/1962/speedread/  A model for the structure of DNA Watson and Crick (1953) proposed a double helix formed by pairing of A-T and C-G in an anti- parallel fashion quite a radical shift from the ideas preceding them that argued that the bases must be on the outside  Wilkins and his colleague Rosalind Franklin provided the key X-ray diffraction patterns that Watson and Crick used, as well as information from many other scientists, to build the definitive model of DNA’s structure. https://www.nobelprize.org/prizes/medicine/1962/speedread/ X-ray diffraction studies of DNA fibres Maurice Wilkins and Rosalind Franklin obtained X-ray diffraction patterns that suggested a helical structure Maurice Wilkins showed a diffraction pattern of DNA at a scientific meeting in Naples in 1951  X-ray diffraction studies of DNA fibres 23 & 24 February 1953 Nearly home Rosalind E Franklin (R.E.F) R.E.F. is at last making the correct A B connection between structures A and B Aaron Klug  J. Michael Creeth In his PhD thesis of 1947, Creeth correctly predicted the DNA molecule comprised two chains, each with a phosphate-sugar backbone on the outside and hydrogen bonded bases on the inside. https://theconversation.com/the-forgotten-scientist-who-paved-the-way-for-the-discovery-of-dnas-structure-86978 The DNA Double Helix Consists of Two Complementary and Antiparallel Strands 10  The DNA Double Helix Consists of Two Complementary and Antiparallel Strands Watson and Crick’s model of the secondary structure of DNA is a fairly simple structure It is composed of four kinds of nucleotides, joined by covalent phosphodiester bonds in a polynucleotide chain Each polynucleotide chain has a sugar-phosphate backbone, consisting of alternating sugar and phosphate groups Two polynucleotide chains come together to form a right-handed anti- parallel double helix 11 DNA Nucleotides ▪ DNA (and RNA) molecules are made from nucleotide building blocks ▪ Nucleotides comprise: ▪ a base, ▪ a sugar, and ▪ a phosphate deoxyadenosine 5’-monophosphate (dAMP) DNA Nucleotides Four nitrogenous bases; two purines (A or G) & two pyrimidines (C or T) 5’-phosphate RNA has ribose (2’OH) in place 3’-hydroxy of deoxyribose DNA Nucleotides (ring) carbons in the carbons or nitrogens (ring sugar are numbered vertices) in the base are 1’-5’ numbered 1-whatever phospho-ester bond glycosidic bond phospho-ester bond nucleosides and nucleotides ▪ Base plus sugar = nucleoside ▪ Nucleosides are named for bases: base nucleoside adenine adenosine cytosine cytidine guanine guanosine thymine thymidine uracil uridine ▪ Nucleoside plus phosphate = nucleotide DNA Nucleotides    The phosphate closest to the sugar is the alpha () phosphate The next closest is the beta () phosphate The furthest away is the gamma () phosphate Only the  phosphate is incorporated into polynucleotides Two Types of DNA Bases ▪ Purines (adenine or guanine) have a double ring 17 Two Types of DNA Bases ▪ Pyrimidines (cytosine, thymine, or uracil) have a single ring 18 You should know and/or be able to recognize the structures of the 5 bases/nucleosides/nucleotides singly or in combination purine pyrimidine purine pyrimidine pyrimidine Complementary DNA Nucleotide Pairing The two polynucleotide chains of a double helix form a stable structure that follows two rules: 1. The bases of one strand are complementary to the bases in the other strand, i.e., they form hydrogen bonds with one another. ▪ A pairs with T, and G pairs with C ▪ A = T base pairs have the same dimensions as G ≡ C base pairs 20 Complementary DNA Nucleotide Pairing The two polynucleotide chains of a double helix form a stable structure that follows two rules: 2. The two chains are antiparallel with respect to their 5 and 3 ends 21  Ball-and-stick diagram of DNA double helix 3D structure of B-DNA ▪ sugar-phosphates form the backbone of molecule held together by phosphodiester linkages via 5' phosphate and 3'hydroxy groups ▪ sugar phosphate backbones run antiparallel to each other  Ball-and-stick diagram of DNA double helix 3D structure of B-DNA The chemical basis of the pairing is the formation of stable hydrogen (H) bonds between the bases on the antiparallel strands ▪ Complementary base pairing aligns one purine with one pyrimidine ▪ “A” pairs specifically with “T” forming two H-bonds ▪ “C” pairs with “G” forming three H- bonds ▪ The dimensions of the base pairs ~11Å are ~identical 3D structure of B-DNA The Twisting Double Helix The DNA double helix has an axis of helical symmetry, an imaginary line that passes lengthwise through the core of the helix 25 The Twisting Double Helix The diameter of the molecule is 20Å (2 nm), where 1Å is 10−10 m The diameter results from the fact that each complementary base pair (A and T or G and C) is ~11Å wide; each complementary nucleotide pair is ~ 20Å wide. 26 Nucleotide Base Stacking Nucleotide base pairs are spaced along the DNA duplex at intervals of 3.4Å This tight packing leads to base stacking, the offsetting of adjacent base pairs so that the base pairs are essentially parallel to one another This leads to a twist in the double helix 27 Major and Minor Grooves Base-pair stacking creates gaps between the sugar-phosphate backbones that partially expose the nucleotides The major groove, approximately 12Å wide, alternates with the minor groove, approximately 6Å wide (these dimensions apply only to B-DNA) 28 The major and minor grooves are regions where DNA binding proteins can make direct and specific contact (H-bonds) with nucleotides DNA binding proteins also interact with DNA through electrostatic interactions with the charged phosphates 29 Watson and Crick proposed a model structure for B-DNA. Is it the real structure? A more sophisticated test is to solve the structure of a DNA molecule by X-ray crystallography and to ask: is the structure the same or different from the structure proposed by Watson & Crick? X-ray crystallography is used to determine the precise position of atoms in a molecule. As the name suggests, crystals must be used. The pictures obtained by Rosalind Franklin were based on fibres not crystals. Compare the structures shown in these two drawings. Are they the same? If not, what differences do you see? 31 Dimensions, complementarity, and handedness all seem the same 32 Base-pairs are not “flat”, and they are sometimes slightly “twisted” 33 Watson & Crick’s model holds up very well compared with the real structure of DNA. As long as we remember that there are slight structural “perturbations”, we can speak of the DNA structure in terms of the way that Watson & Crick described it. The complementary base-paired structure of DNA means that: ▪ DNA can be “melted” (strands separated) and the strands can only go back together (reannealed) in one way (DNA hybridization) ▪ DNA can be copied into DNA (duplicated) faithfully because each strand can be copied but only the correct complementary sequence can be made ▪ DNA can be copied into RNA because RNA is also composed of bases that must match those in the original sequence 35 The complementary base-paired structure of DNA means that: ▪ DNA can be over- or under-wound causing torsional stress providing potential energy for strand separation ▪ The over- or underwinding of (essentially) closed DNA molecules results in supercoiling 36  Supercoiling is a result of untwisting or over-twisting the double helix o n untwist e ½ turn t u r n If you untwist the molecule, you introduce fewer turns per unit length (and the molecule is no longer thermodynamically comfortable)  Supercoiling is a result of untwisting or over-twisting the double helix o Over twist n e 1½ turn t u r n If you over twist the strands, you introduce more turns per unit length (again, the molecule is no longer thermodynamically comfortable) Supercoiling ▪ Supercoiling relieves the stress that is introduced as a result of over- or under- twisting DNA ▪ It is particularly evident in covalently closed circular molecules of DNA ▪ The relaxed circle is the least twisted ▪ Supercoiling also compacts DNA 39 Uncomfortable molecules try to become more comfortable! Thermodynamically unstable molecules try to become more stable! It facilitates the organization of large DNA molecules into smaller spaces Positive supercoiling twists the DNA so that it is over-rotated Negative supercoiling twists the DNA so that it is under-rotated ▪ Negative supercoils can be a benefit because they allow DNA to ‘melt’ – an important first step for replication and for transcription NOTE: The role of supercoiling in packaging DNA into nucleosomes, and the consequences of that for eukaryotic gene expression will be presented in later classes. 41 Supercoiling & Topoisomerases Supercoiling is regulated by enzymes called Topoisomerases The supercoiled state of DNA can be changed in two ways: ▪ One of the two strands can be cut, it can be rotated around the other strand, and it can be re-joined to its partner ▪ Both strands can be cut, both are rotated, and then they are re-joined Re-joining must always respect the 5’->3’ polarity of a DNA strand Topoisomerases are important enzymes both from a disease and from a clinical perspective 42 Topoisomerases and Disease Scleroderma ▪ an autoimmune disease-causing hardening of skin and organs ▪ auto-antibodies (Anti-Scl-70) are against topoisomerase ▪ systemic form is untreatable and fatal Lupus ▪ an autoimmune disease ▪ there is evidence that topoisomerase I is a target in some patients Cancer overexpression of topoisomerase II alpha has been reported in colorectal tumors and Hodgkin’s disease (may be important in allowing growth) Topoisomerases as clinical targets Antibiotics some antibiotics (ciprofloxacin, novobiocin) function by inhibiting DNA gyrase ( a type of topoisomerase) which is only found in prokaryotes (e.g., bacteria) Chemotherapeutic agents some anti-cancer drugs inhibit eukaryotic type II topoisomerases and, therefore, selectively kill rapidly dividing cells 44 A little bit more about “Real” DNA The structure of “real” DNA can deviate from a perfect Watson-Crick double helix In addition to what we mentioned earlier: sequence context different base sequences have a greater or lesser ability to rotate or twist causing distortions of the helix, e.g. ▪ A “run of adenines” in one strand can have significant ring-stacking overlap giving a localized rigidity ▪ alternating purines and pyrimidines are more flexible and can twist more 45 A little bit more about “Real” DNA There are TWO major structural forms of DNA A-DNA Recall that Rosalind Franklin saw two forms of fibre structure A-DNA less hydrated form of DNA B-DNA has a significant role when RNA strands form double helices 46 A little bit more about “Real” DNA The B-form is more hydrated This is due to the presence of a “spine” of hydrogen-bonded water molecules in the minor groove McDermott, M.L., Vanselous, H., Corcelli, S.A. and Petersen, P.B., 2017. DNA’s chiral spine of hydration. ACS central science, 3 (7), pp.708-714. 47 A little bit more about “Real” DNA When the first crystal structure of a DNA Z-DNA molecule was obtained, it had a most unexpected structure… It was Left-handed…!!! B-DNA This molecular structure was determined from a crystal of: 5’- GCGCGC -3’ 3’- CGCGCG -5’ 48 Z-DNA In the GCGCGC crystal, bases were flipped from the anti to the syn configuration resulting in a left-handed double helix Possibly important in control of (some) gene expression 49 The anti and syn configuration ▪ The glycosidic bond has rotational freedom ▪ The base can be positioned over the sugar (syn) or away from it (anti) anti syn 50 The B-structure is by far the most common found in DNA in cells 51  Take home message DNA has hidden depths; it is not just the simple double helix that you might have thought it was! and many of these alternate forms/structures may have real biological relevance However, The B-form is still thought to be by far the most prevalent form that DNA takes in cells Therefore, it is the form will we concern ourselves with in this course 52  So, for our purposes we will think of DNA as a nice regular Watson and Crick type of double helix existing in the B-form 53