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Structure of DNA
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Structure of dna

  1. 1. DNA A molecule that contains the instructions an organism needs to develop, live and reproduce. These instructions are found inside every cell and are passed down from their parents to their children. Nearly every cell in the persons body has the same DNA
  2. 2. Made up of molecules called nucleotides. Each molecule contains a phosphate group , a sugar group and a nitrogen base. These nitrogen bases are adenine(A),guanine(G),thymine (T)and cytosine(C). The order of these bases is what determines the DNA’s instructions or genetic codes. COMPOSITION OF DNA
  3. 3. NUCLEIC ACIDS  Nucleic acids are polymers  Monomer-----nucleotides Components:-  Nitrogenous bases  Purines Pyrimidines  Sugars Ribose Deoxyribose  Phosphates + nucleosides=nucleotides
  4. 4. NUCLEIC ACIDS  Nucleic Acids, are naturally occurring chemical compound that is capable of being broken down to yield phosphoric acid, sugars, and a mixture of organic bases (purines and pyrimidines).  Nucleic acids are the main information-carrying molecules of the cell, and, by directing the process of protein synthesis, they determine the inherited characteristics of every living thing.  The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).  DNA is the master blueprint for life and constitutes the genetic material in all free-living organisms and most viruses.  RNA is the genetic material of certain viruses, but it is also found in all living cells, where it plays an important role in certain processes such as the making of proteins.
  5. 5.  Nucleic acids are polynucleotide—that is, long chainlike molecules composed of a series of nearly identical building blocks called nucleotides.  Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar, which is in turn attached to a phosphate group. Each nucleic acid contains four of five possible nitrogen containing bases: adenine (A), guanine (G), cytosine (C), thymine (T)and Uracil(U).  A and G are categorized as purines, and C, T, and U are collectively called pyrimidines. All nucleic acids contain the bases A, C, and G; T, however, T is found only in DNA, while U is found in RNA.  The pentose sugar in DNA (2′-deoxyribose) differs from the sugar in RNA (ribose) by the absence of a hydroxyl group (−OH) on the 2′ carbon of the sugar ring. Without an attached phosphate group, the sugar attached to one of the bases is known as a nucleoside.  The phosphate group connects successive sugar residues by bridging the 5′- hydroxyl group on one sugar to the 3′-hydroxyl group of the next sugar in the chain. These nucleoside linkages are called phosphodiester bonds and are the same in RNA and DNA.
  6. 6. THE SUGARS (in DNA) (in RNA)  DEOXYRIBOSE  Deoxyribose is a pentose sugar of RNA with five sugars . Four out of five carbon atoms plus an oxygen atom forms a five membered ring  The fifth carbon is outside the group and forms the part of the – CH2 group.  Deoxyribose has only two (-OH) groups (on carbons 3’ and 5’) and thus can only form two deoxyribonucleotides , the 3’ and 5’ phosphate derivatives.  RIBOSE  Ribose is a pentose sugar of RNA with five carbons.  It has an identical structure to DNA except there is a (-OH) group instead of hydrogen on carbon atom 2’.  Ribose has free (-OH) groups on carbons 2’, 3’ and 5’.  The phosphate can attach to any of these three positions.
  7. 7. THE BASES  The main nitrogenous bases present in DNA and RNA are purines and pyrimidines.  PURINES  A series of heterocyclic compounds that are variously substituted in nature are known also as purine bases .  They include adenine and guanine as constituents of nucleic acids and many alkaloids. PYRIMIDINES  A heterocyclic compound C4H4N2,that is the basis of several important biochemical substances.  They include cytosine , thymine and uracil as constituents of nucleic acids.
  9. 9. TAUTOMERIC FORMS OF BASES:SOME OF THE POSSIBLE TAUTOMERIC FORMS OF (a)guanine and (b)thymine. Cytosine and adenine can also undergo similar proton shifts. Guanine (enol or lactim form)=Guanine (keto or lactam form) Thymine(enol or lactim form)=Thymine (keto or lactam form).
  10. 10. NUCLEOTIDES-THE BUILDING BLOCKS OF DNA NUCLEOTIDES, any member of a class of organic compounds in which the molecular structure comprises a nitrogen-containing unit (base) linked to a sugar and a phosphate group.  The nucleotides are of great importance to living organisms, as they are the building blocks of nucleic acids, the substances that control all hereditary characteristics.  Several nucleotides are coenzymes; they act with enzymes to speed up (catalyze) biochemical reactions.
  11. 11.  Nucleotides are synthesized from readily available precursors in the cell.  The ribose phosphate portion of both purine and pyrimidine nucleotides is synthesized from glucose via the pentose phosphate pathway.  The six-atom pyrimidine ring is synthesized first and subsequently attached to the ribose phosphate.  The two rings in purines are synthesized while attached to the ribose phosphate during the assembly of adenine or guanine nucleosides.  In both cases the end product is a nucleoside carrying a phosphate attached to the 5′ carbon on the sugar. Finally, a specialized enzyme called a kinase adds two phosphate groups using adenosine triphosphate (ATP) as the phosphate donor to form ribonucleoside triphosphate, the immediate precursor of RNA. For DNA, the 2′-hydroxyl group is removed from the ribonucleoside diphosphate to give deoxyribonucleoside diphosphate. An additional phosphate group from ATP is then added by another kinase to form a deoxyribonucleoside triphosphate, the immediate precursor of DNA.  During normal cell metabolism, RNA is constantly being made and broken down. The purine and pyrimidine residues are reused by several salvage pathways to make more genetic material. Purine is salvaged in the form of the corresponding nucleotide, whereas pyrimidine is salvaged as the nucleoside.
  13. 13. THE SEVEN TORSION ANGLES THAT DETERMINE THE CONFORMATION OF A NUCLEOTIDE UNIT The conformation of a nucleotide unit,as the fig indicates,is specified by the six torsion angles of the sugar – phosphate backbone and the torsion angle describing the orientation of the base around the glycosidic bond (the bond joining the c1’ to the base. It would seem that these seven degrees of freedom per nucleotide would render the polynucleotide very flexible. Yet these torsion angles are subject to a variety of internal constraints that greatly restrict their rotational freedom. The rotation of a base around its glycosidic bond (angle χ) is greatly hindered.
  14. 14. THE STERICALLY ALLOWED ORIENTATIONS OF PURINE AND PYRIMIDINE BASES W.R.T.THEIR ATTACHED RIBOSE UNITS;IN B-DNA,THE NUCLEOTIDE RESIDUES ALL HAVE ANTI CONFORMATIONS Purines residues have two sterically permissible orientations known as the syn-(greek:with) and anti- (greek:against) conformations. Only the anti conformations of pyrimidines is stable,because in the syn conformation ,the sugar residue sterically interferes with the pyrimidine’s C2 substituent. In most double helical nucleic acids, all bases are in the anti conformation. The exception is Z-DNA,in which the alternating purine and pyrimidine residues are anti and syn,respectively (this is the one reason why the repeating unit of Z-DNA is a dinucleotide).
  15. 15. The flexibility of the ribose ring itself is also limited. The vertex angles of a regular pentagon are 108˚,a value quite close to the tetrahedral angle (109.5˚),so one might expect the ribofuranose ring to be nearly flat, however the ring substituents are eclipsed when the ring is planar. To relieve this crowding, which occurs even between hydrogen atoms, the ring puckers i.e. it becomes slightly non-planar. In the great majority of known nucleoside and nucleotide x-ray structures, four of the ring atoms are co-planar to within a few hundredths of an angstrom and the remaining atom is out of this plane by several tenths of an angstrom. The out of plane atom is almost always c2’ and c3’.
  16. 16. The two most common ribose conformations are known as C3’-endo and C2’- endo;”endo” (greek: end on, within) indicates that displaced atom is on the same side of the ring as C5’. The ribose pucker is conformationally important in nucleic acids because it governs the relative orientations of phosphate substituents to each ribose residue. In fact, B-DNA has the C2’-endo conformation, whereas A-DNA is C3’-endo In Z-DNA, the purine nucleotides are all c3’-endo and the pyrimidine nucleotides are C2’-endo.
  17. 17. DNA STRUCTURE DNA is a molecule duplex i.e consists of two chains arranged in a antiparallel manner and with nitrogenous bases facing each other. In a three-dimensional there are three different levels Primary Secondary Tertiary
  18. 18. SUMMARY OF PRIMARY,SECONDARY AND TERTIARY STRUCTURES Primary structure:  Sequence of nucleotide chains. It is in these channels where the genetic information, and because the skeleton is the same for all ,the difference in the information lies in the different sequence of nitrogenous bases. This sequence has a code, which determines an information or otherwise, as the order of the bases. Secondary structure:  It is a double helix structure. Can explain the storage of genetic information and the mechanism of DNA replication. It was postulated by Watson and Crick, based on X-ray diffraction that Franklin and Wilkins had made, and the equivalence of bases Chargaff's postulated, whereby the sum of adenines more guanines is equal to the sum of thymine more cytosine. It is a double strand, right-handed or left-handed, depending on the DNA. Both chains are complementary, as adenine and guanine in a chain are joined, respectively , to thymine and cytosine on the other. Both chains are antiparallel, then the 3 'end of one faces the 5' end of the counterpart.  There are three models of DNA. The DNA of type B is the most abundant and is discovered by Watson and Crick.
  19. 19. Tertiary structure:  Refers to how DNA is stored in a confined space to form the chromosomes. Varies depending on whether the organisms prokaryotes and eukaryotes:  In prokaryotes the DNA is folded like a super-helix, usually in circular shape and associated with a small amount of protein.  In eukaryotes, since the amount of DNA from each chromosome is very large, the packing must be more complex and compact, this requires the presence of proteins such as histones and other proteins of non- histone nature (protamines).
  20. 20.  PRIMARY STRUCTURE  A single DNA chain is a long thread like molecule made up of a large no. of deoxyribonucleotides.  The backbone of primary structure consists of deoxyribose linked by phospho-diester bridges.  The phospho-diester bond are formed between 3’- and 5’-of the successive sugar molecules.  The 3’OH group of deoxy-pentose of one nucleotide is joined to 5’OH group of deoxy-pentose of the adjacent nucleotide through a phosphate group.  This way a long unbranched chain is formed which has the polarity a 5’end and a 3’end are free(phosphate groups are free without the phospho-diester linkage not attached to other nucleotides). LEVELS OF STRUCTURE OF DNA
  22. 22.  Two antiparallel polynucleotide chains wound around the same axis.  Sugar phosphate chains wound around the periphery.  Bases A,T,G and C occupy the core , forming A:T and G:C Watson-Crick base pairs.  The DNA double helix is held together mainly by- Hydrogen Bonds. SECONDARY STRUCTURE OF DNA
  23. 23. HYDROGEN BOND  A chemical bond in which a hydrogen atom of one molecule is attracted to an electronegative atom of another molecule (especially a nitrogen, oxygen or fluorine atom).
  24. 24. EVENTS LEADING TO THE DNA STRUCTURE  In 1953,James Watson and Francis Crick discovered the double helical structure of DNA.  The scientific framework for their breakthrough was provided by other scientists including -Linus Pauling -Rosalind Franklin -Erwin Chargaff
  26. 26. X-RAY CRYSTALLOGRAPHY  X-Ray diffraction study of DNA by W.T.Astbury (1940s) indicated that DNA is a polynucleotide chain , where successive nucleotides occur at 3.4 A.  Franklin (1952) observed DNA to be a helix.  Wilkins and Franklin (1953) obtained very fine x-ray diffraction pictures of DNA which were immediately made available to Watson and Crick.  Piecing together all the previous information ,Watson and Crick(1953) came to the conclusion that DNA was made of two anti-parallel helical chains held together by hydrogen bonds created between their nitrogen bases.
  27. 27. ERWIN CHARGAFF’S EXPERIMENT  It was assumed the four bases; A,G,C and T were in a repeating tetranucleotide configuration.  Therefore , there should be the same amount of A,G,C and T in any molecule of DNA from any source.  Chargaff carefully determined the exact percentages of nucleotides in DNA from several sources.  %A=%T and %G=%C.  However %AT did not equal to %GC.  This observation became known as Chargaff Rule.
  29. 29. BASE PAIRING It is a pairing formed in the DNA double helix between purine of one strand and pyrimidine of the second strand. Base pairing is specific with adenine lying opposite thymine and cytosine occurring opposite guanine. It can accommodate neither two purines , nor two pyrimidines.
  30. 30.  According to Watson and Crick, a DNA molecule consists of two polynucleotide chains wrapped helically around each other,with the sugar phosphate chains on the outside and purines and pyrimidines on the inside of the helix.  The two chains are spirally coiled around a common axis in a regular manner to form a double helix.  The double helix is of constant diameter of 2 nanometers(nm) or 20 angstroms and has a major groove, about 20 angstroms wide and the minor groove about 12 angstroms wide alternately.  One complete spiral helix is 34 angstroms long and has 10 base pairs.  The bases face the interior of the double helix and are stacked 3.4 angstroms apart. WATSON AND CRICKS MODEL OF DNA
  31. 31.  The sugar phosphate component forms the backbone on the outside.  The two strands run strand has phosphodiester linkage in the 3’ -5’ direction, while the other strand has phosphodiester linkage in the 5’-3’ direction.  The helix is generally right handed that is it runs clockwise looking along the helix axis.  The two strands are held together by hydrogen bonds between specific base pairs of purines and pyrimidines. The hydrogen bond between purines and pyrimidines are such that adenine can bond only to thymine by two hydrogen bonds and guanine can bond only to cytosine by three hydrogen bonds.  The specificity of the kind of hydrogen bonds that can be formed assures that for every adenine in one chain there will be thymine in the other and for every guanine in one chain there will be cytosine in the other. Thus the two chains are complementary to each other.
  32. 32. DOUBLE HELIX-RIGHT HANDED AND LEFT HANDED COILING  The double helix is a spiral right- handed, that is, each of the nucleotide chains turn right, this can be verified if we look at the segment (a), where the threads move upwards and eventually turn right .  If the two strands rotate clockwise it is said that the double helix is right- handed, and if they turn towards left, left-handed (this form may appear in helices alternatively because of conformational changes in DNA).  But the most common conformation adopted by the DNA double helix is right-handed, turning every couple of bases on the previous approximately 36 º.
  33. 33.  When the two DNA strands are rolled over each other (either left or right), cracks are formed between a thread and the other, exposing the sides of the nitrogenous bases inside.  In the most common conformation DNA adopts, because of the angles between the sugars of both strands of each pair of nitrogenous bases, appears two types of cracks around the surface of the double helix: one, the cleft or major groove, which is 22 Å (2.2 nm) wide, and the other, the minor groove, which is 12 Å (1.2 nm) wide.  The major groove is wider than the minor groove in DNA;and many sequence specific proteins interact in the major groove.  The N7 and C6 groups of purines and the C4 and C5 groups of pyrimidines,face into the major groove.  Thus, they can make specific contacts with amino acids in DNA binding proteins.  Thus specific amino acids serve as H-bond donors and acceptors to form H-bonds with specific nucleotides in DNA.  H-bond donors and acceptors are also in the minor groove and indeed some proteins bind specifically ,in the minor groove.  Base pairs stack with some rotation between them.
  34. 34. MAJOR HELICAL CONFORMATIONS OF DNA Most of the biologically active DNA exists in Watson-Crick form . This is the B-form of the DNA.The double helix is able ton assume other forms depending upon varying environmental conditions.  6 MORPHOLOGICAL FORMS OF DNA  A,B,C,D,E and Z.  A-DNA: Right handed helix with 11 base pair turns . It is the dehydrated form which occurs in the environment richer in Na+ and less of water.  B-DNA: Watson and Crick’s model of DNA is the common form of DNA found in organisms . It is a right handed helix with each turn of spiral having 10 base pairs . It occurs under salt concentration and high degree of hydration.  C-DNA: Right handed helix with 9 base pairs per turn.  D-DNA: Right handed helix with 8 base pairs per turn.  E-DNA: Form adapted by synthetic DNA lacking guanine . There are 7½ base pairs per turn.  C-DNA and E-DNA are seen under special environmental conditions and have slightly different conformation so do not occur in vivo.  Z-DNA: Left handed helix with zigzag back of sugar phosphate residues and 12 base pair per turn of helix . It is the skinniest DNA, with only one groove and is stabilized by high salt concentration.
  35. 35. 3 MAJOR FORMS OF DNA
  36. 36. CONFORMATIONS OF A,B and Z DNA DNA exists in many conformations. However, in living organisms have only been observed conformations A-DNA, B-DNA and Z-DNA. The conformation DNA adopts depends on its sequence, the amount and direction of super coiling that show the presence of chemical modifications on the bases and conditions of the solution, such as the concentration of ions of metals and polyamines. In the three conformations, the form "B" is the most common conditions in the cells. The two DNA double helices alternatives differ in their geometry and dimensions.
  37. 37. Under dehydrating conditions-DNA undergoes a reversible conformational change to A –DNA. A-DNA ,which forms a wider and flatter right –handed helix than does B-DNA. A-DNA has 11.6 bp per turn and a pitch of 34 Angstroms, which gives it an axial hole. A-DNA’s most striking feature ,however, is that the planes of its base pairs are tilted 20 degree w.r.t the helix axis. Since the axis does not pass through its base pairs, A –DNA has a deep major groove and a very shallow minor groove ;it can be described as a flat ribbon wound around a 6-Angstroms diameter cylindrical hole.
  38. 38.  In B DNA ,the bases occupy the core of the helix while the sugar phosphate backbones wind around the outside, forming the major and minor grooves. Only the edges of the base pairs are exposed to the solvent.  The “ideal” B DNA helix has 10 base pairs (bp) per turn (a helical turn of36 degree per bp) and, since the aromatic bases have van der waal’s thickness of 3.4 Angstroms and are partially stacked on each other, the helix has a pitch (rise per turn )of 34 Angstroms.  The helix of Z DNA has 12 BASE pairs per turn, a pitch of 45 Angstroms ,a deep minor groove, and no discernible major groove.  Z-DNA, therefore resembles a left- handed drill bit in appearance.
  39. 39. SIMILARITIES BETWEEN Z-DNA and B-DNA.  Both are double helical.  In both DNAs ,two polynucleotide strands of double helix are antiparallel.  Both forms exhibit G triple bond C PAIRING. DIFFERENCES BETWEEN Z-DNA and B-DNA.  Z-DNA has left-handed helical sense, while B-DNA has right handed helicalsenses  The phosphate back bone of Z-DNA follows a zigzag course ,while in B-DNA this backbone is regular.  In Z-DNA , the adjacent sugar residues have opposite orientation, while in B- DNA , they have same orientation. Due to this, the repeating unit is a dinucleotide in Z-DNA as against a mononucleotide unit in B-DNA.  In Z-DNA , one complete helix(i.e. a twist through 360˚ has twelve base pairs or six repeating dinucleotide units, while in B-DNA one complete helix has only 10 base pairs or 10 repeating units.  In Z-DNA, one complete turn of helix is 45˚ long, while in B-DNA it is 34 Angstroms long.
  40. 40. DENATURING AND ANNEALING OF DNA The DNA double strand can be denatured if heated (95 C) or treated with chemicals.  AT regions denatures first (2H bonds)  GC regions denatures last (3H bonds) DNA denaturation is a reversible process , as DNA strands can be re- annealed if cooled. This process can be monitored using the hyperchromicity (melting profile). Hyperchromicity:  It is used to moniter DNA denaturation and annealing.  It is based on the fact that single stranded(SS) DNA gives higher absorption reading than double stranded (DS) at wavelength 260 nm.
  41. 41. DENATURATION  The strands of the DNA double helix are held together by hydrogen bonding interactions between the complementary base pairs. Heating DNA in solution easily breaks these hydrogen bonds, allowing the two strands to separate—a process called denaturation or melting.  The two strands may reassociate when the solution cools, reforming the starting DNA duplex—a process called renaturation or hybridization.  These processes form the basis of many important techniques for manipulating DNA. For example, a short piece of DNA called an oligonucleotide can be used to test whether a very long DNA sequence has the complementary sequence of the oligonucleotide embedded within it.  Using hybridization, a single-stranded DNA molecule can capture complementary sequences from any source. Single strands from RNA can also reassociate. DNA and RNA single strands can form hybrid molecules that are even more stable than double-stranded DNA.  These molecules form the basis of a technique that is used to purify and characterize messenger RNA (mRNA) molecules corresponding to single genes.
  42. 42. ULTRAVIOLET ABSORBPTION • DNA melting and reassociation can be monitored by measuring the absorption of ultraviolet (UV) light at a wavelength of 260 nanometers (billionths of a metre). When DNA is in a double-stranded conformation, absorption is fairly weak, but when DNA is single-stranded, the unstacking of the bases leads to an enhancement of absorption called hyperchromicity(melting profile). Therefore, the extent to which DNA is single-stranded or double-stranded can be determined by monitoring UV absorption.
  44. 44. BOUYANT DENSITY OF DNA It is the density of the solution at which the DNA feels no net force during centrifugation is called its buoyant density. This is the density in the density gradient where that particular DNA molecule will form bands as it stops going up or down.
  45. 45. • Under constant conditions (usually 25˚C in ceasium chloride at neutral pH ) the buoyant density of DNA is related to the GC content. • The fractionation of Phaseolus aerus DNA has a buoyant density of 1.695g/cm³ and that of E.coli DNA has a buoyant density of 1.710 g/cm³in ceasium chloride. • Most nuclear DNAs from higher plants have buoyant density within the range 1.69-1.71 g/cm³. • However the presence of 5-metyl-cytosine serves to reduce the density slightly , thereby giving rise to an under estimate of the GC content. • In general, 1% methylation decreases the buoyant density by 1 mg/cm³. • Certain sequences of bases may also distort the relationship between the base composition and buoyant density. • Furthermore , ssDNA is denser than dsDNA of similar base composition by approximately 0.015 g/cm³ and under alkaline conditions the density is increased by 0.06 g/cm³
  46. 46. FACTORS AFFECTING BUOYANT DENSITY • Buoyant Density of DNA depends on the following factors: • Nature of the ceasium chloride, • Presence of heavy metals or DNA binding dyes, • The pH, and • The temperature.