DNA structure, the bonds involved and it seperation

DNA structure, and the bonds that stabilizes it. The structural components, units and the proteins involved. Types of DNA and its separation methods. Chargaffs rule and its application

1. DNA STRUCTURE

2. Outline Terms used. Polarity Bonds present Proteins involved Properties of nucleosides and nucleotides Structure of DNA Watson Crick model Types of DNA DNA sequencing

3. Terminologies…. Nitrogenous base. Nitrogen containing. Free pyrimidines and purines are weakly basic compounds and thus are called bases Sugar 2-Deoxy D-ribose pentose sugar

4. Terminologies…. Nucleoside nitrogenous (nitrogen-containing) base a pentose Nucleoside with Purine base have suffix –sine Pyrimidine base have suffix –dine Nucleotide nitrogenous (nitrogen-containing) base a pentose a phosphate Nucleic acid Linear polymers of nucleotides

5. Introns Non-coding, intervening , silent areas Exons Coding, expressed regions Cistrons Unit of gene expression, biochemical counterpart of gene Gene A segment of a DNA molecule that contains the information required for the synthesis of a functional biological product, whether protein or RNA.

6. Bases involved Why thymine in DNA???

7. Deoxyribonucleosides Adenine Deoxyribose Deoxy -adenosine (d-adenosine) Guanine Deoxyribose Deoxy-guanosine (d- guanosine ) Cytosine Deoxyribose Deoxy-cytidine (d- cytidine ) Thymine Deoxyribose Deoxy-thymidine (d- thymidine )

8. Polarity In DNA the base sequence is always written in 5 I end to 3 I end direction By definition, the 5 end lacks a nucleotide at the 5 position and the 3 end lacks a nucleotide at the 3 position. 5 I -OH and 3 I- OH ends. The 5 to 3 orientation of a strand of nucleic acid refers to the ends of the strand.

9. Bonds involved… N-9 of a purine or N-1 of a pyrimidine is attached to C-1 of the sugar The base lies above the plane of sugar when the structure is written in the standard orientation; The configuration of the N- glycosidic linkage is β .

10. Bonds involved… The successive nucleotides of DNA covalently linked through phosphate-group “bridges” 5-phosphate group of one nucleotide unit is joined to the 3-hydroxyl group of the next nucleotide 3 I- to 5 I Phosphodiester linkage

11. Covalent backbones of nucleic acids consist of alternating phosphate and pentose residues, Nitrogenous bases as side groups joined at regular intervals. Linkages can be cleaved hydrolytically by chemicals or enzymatically by family of Nucleases.

12. Comments… The covalent backbone of DNA and RNA is subject to slow, non-enzymatic hydrolysis of the phosphodiester bonds. In the test tube, RNA is hydrolyzed rapidly under alkaline conditions but DNA is not.

13. Hydrogen bonds Involving the amino and carbonyl groups are the most important mode of interaction between two complementary strands of nucleic acid. Required for specificity of base pairing

14. Other bonds… The stacking also involves a combination of van der Waals and dipole-dipole interactions between the bases. Base stacking helps to minimize contact of the bases with water, and Base -stacking interactions are very important in stabilizing the three dimensional structure of nucleic acids Base stacking in DNA is also favored by the conformations of the relatively rigid five- membered rings of the backbone sugars. The sugar rigidity affects both the single-stranded and the double-helical forms.

15. Properties of nucleosides and nucleotides Physical properties Charged phosphate groups provide interaction. Electron delocalization among atoms in the ring gives most of the bonds partial double-bond character. Strong absorption at wavelengths near 260 nm. Purines have higher molar extinction coefficient.

16. Structural properties Sugar puckering Displacement of 2 and 3 carbon atom above the plane of C1-O4-C4 By convention above is the direction in which the base and C5 I project from the ring and is termed the endo face of the pentose. If C2 is above it is called C2 endo If C3 is above it is called C3 endo Orientation of glycosidic bond and the distance of the phosphate bond changes. DNA: C2 endo and RNA C3 endo .

17. Anti- & Syn - conformation… Nucleosides are planar. Still free rotation of glycosidic bond is possible. In purines H8 above sugar- anti conformation H8 below and far with bulk og purine ring above sugar- syn In pyrimidines in pyrimidines H6 is above sugar- anti conformation O2 is above sugar- syn conformation

18. Comments… Purines favour anti conformation.. Interconvert between the two Guanine 5 I nucleotides is always an exception Favours syn conformation. Importance Defines the handedness of the DNA helix. Anti conformation- right handed helix Syn - conformation- left handed helix DNA having more of G will have left handed helix

19. Handedness If you look up through the bottom of a helix along the central axis and the helix spirals away from you in a clockwise direction it is a right-handed helix. If it spirals away from you in a counter-clockwise direction, it is a left handed helix.

20. Proteins involved… Histones Genes for histones seem to have no introns Nucleosome Dna wraps twice around histone octamer

21. Nucleosome

22. Condensed Chromosome Structures Are Maintained by…. A third major class of chromatin proteins SMC proteins ( structural maintenance of chromosomes). Proteins in the SMC family are found in all types of organisms, from bacteria to humans. Eukaryotes have two major types, cohesins and condensins . Cohesins linking together sister chromatids Condensins – condensation of chromosomes as cells enter mitosis

23. DNA & its history… DNA is in nucleus.. Storage of inorganic phosphate.. It carries genetic information It is passed to daughter cell… Then Experiments to elucidate the structure..

24. DNA helix in history…. DNA was first isolated and characterized by Friedrich Miescher in 1868. He called the phosphorus-containing substance “ nuclein .” Alfred D. Hershey and Martha Chase, in which they studied the infection of bacterial cells by a virus ( bacteriophage ) with radioactively labeled DNA or protein, removed any remaining doubt that DNA, not protein, carried the genetic information

25. 1951—Rosalind Franklin—X-ray crystallography Chargaff—Chargaff’s rules. Ratio of nitrogenous bases in DNA. Complimentary bases. Watson & Crick--1953

26. Erwin Chargaff

27. Chargaff’s conclusions Base composition of DNA varies from one species to another. DNA specimens isolated from different tissues of the same species have the same base composition. The base composition of DNA in a given species does not change with an organism’s age, nutritional state, or changing environment. In all cellular DNAs, regardless of the species, Number of adenosine residues is equal to thymidine residues (that Number of guanosine residues is equal to cytidine residues Sum of the purine residues equals pyrimidine residues

28. Watson Crick model

29. Two helical polynucleotide chains are coiled around a common axis. The chains run in opposite directions. The sugar-phosphate backbones are on the outside and, therefore, Purine and pyrimidine bases lie on the inside of the helix. The bases are nearly perpendicular to the helix axis, and adjacent bases are separated by 3.4 Å. The helical structure repeats every 34 Å, so there are 10 bases (= 34 Å per repeat/3.4 Å per base) per turn of helix.

30. There is a rotation of 36 degrees per base (360 degrees per full turn/10 bases per turn). The diameter of the helix is 20 Å. The complimentary nitrogenous bases form hydrogen bonds between the strands. A is complimentary to T and G is complimentary to C.

31. B- DNA, right handed, 10 base pairs per turn. Two chains coiled around a common axis: Axis of symmetry. Pairs in an anti- parallel manner i.e. 5′- end pairs with 3′- end of other strand. Phosphate molecules are hydrophillic , whereas bases forms the hydrophobic molecules.

32. Spatial arrangement of two strands creates Major (wide) & minor (narrow) Groove. Bases are perpendicular to axis, sugars are at right angle to those of bases. Diameter of helix is 20nm, adjacent bases separated by 3.4Ǻ. One complete turn of 10 BP occurs at interval of 34Ǻ.

33. Circular DNA molecules Each chromosome in the nucleus of a eukaryote contains one long linear molecule of dsDNA . Eukaryotes have closed circular DNA molecules in their mitochondria. A prokaryotic organism contains a single, double-stranded, supercoiled , circular chromosome. Each prokaryotic chromosome is associated with histone -like proteins and RNA that can condense the DNA to form a nucleoid . Species of bacteria also contain small, circular, extrachromosomal DNA molecules called plasmids.

34. Other types of DNA Bent DNA 4-6 adenines separated by 10 bp . Minor grooves compressed Interaction with proteins & enzymes Cruciform DNA Disruption of hydrogen bonds between complimentary bp Formation of intra strand hydrogen bonds. Formation of hairpin

35. Other types of DNA Triple stranded DNA Polynucleotides of Poly ( dA ) & poly ( dT ) Hoogsteen triple helix TAT, CGC triplet bp Four stranded DNA Rich in guanine nucleotides. G-quartets. Guanine with hoogsteen hydrogen bonds. Existence invivo not proven

36. Slipped DNA Direct repeat symmetry Formation of two single stranded loops. Fragile X syndrome typical example of triple repeat sequence. Frame shift mutation explained on this basis.

37. Triple stranded DNA

38. Melting temperature Temperature at which one half of the helical structure is lost is defined as the melting temperature (T m ). The loss of helical structure in DNA, called denaturation , can be monitored by measuring its absorbance at 260 nm Ss DNA has a higher relative absorbance at this wavelength than does ds DNA . Complementary DNA strands can reform the double helix by the process called renaturation (or reannealing ).

39. Importance of T m Critical importance in any technique that relies on complementary base pairing Designing PCR primers Southern blots Northern blots Colony hybridization

40. Factors Affecting T m G-C content of sample Presence of intercalating agents (anything that disrupts H-bonds or base stacking) Salt concentration pH Length

41. DNA packaging

42. DNA Sequencing 1. Sanger dideoxynucleotide chain termination method A. Manual method B. Automated method 2. Chemical cleavage method ( Maxam and Gilbert method) Not used nowadays Use of the technique Provides the order of the nucleotides in a given DNA

43. Sanger Method Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP Separate by gel electrophoresis Read DNA sequence

44. Requirements for Sanger Method DNA to be sequenced must be in single strand form. The region to be sequenced must be 3 I flanked by known sequence. Reagents needed: A primer complementary to the known region to start and direct chain synthesis. (15-30 nucleotides in length) DNA polymerase. 4 deoxynucleotide triphosphates ( dNTPs ). 4 dideoxynucleotide triphosphates ( ddNTPs )

45. Take home message… All life on earth use nucleic acid to store genetic information. Except some viruses(RNA) it is stored in DNA. So why DNA??? Chemical stability.. Store vast genetic information And most important!!!!! Encode this vast genetic information with simple four letter code(A,G,C &T)

46. References.. Text Book Of Biochemistry With Clinical Correlation By Thomas Devlin 7 th Edition Lehninger Principles Of Biochemistry. 5th Edition Lippincott's Illustrated Reviews: Biochemistry 4 th Edition Stryer's Biochemistry - 5 th edition Marks’ Basic Medical Biochemistry: A Clinical Approach, 2nd Edition Textbook Of Biochemistry DM Vasudevan 6 th Edition