Se ha denunciado esta presentación.
Se está descargando tu SlideShare. ×

Basics of DNA Structure

Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Anuncio
Cargando en…3
×

Eche un vistazo a continuación

1 de 31 Anuncio

Basics of DNA Structure

Descargar para leer sin conexión

The presentation covers all details of the DNA structure for an easy understanding of Molecular biology students. It covers the details of DNA structure, its bonds as well as the different conformations.

The presentation covers all details of the DNA structure for an easy understanding of Molecular biology students. It covers the details of DNA structure, its bonds as well as the different conformations.

Anuncio
Anuncio

Más Contenido Relacionado

Presentaciones para usted (20)

Similares a Basics of DNA Structure (20)

Anuncio

Más reciente (20)

Anuncio

Basics of DNA Structure

  1. 1. DNA Structure Dr. Riddhi Datta Department of Botany Dr. A.P.J. Abdul Kalam Government College West Bengal State University cbcs Botany Core viii
  2. 2. Let’s make a DNA molecule! © Dr. Riddhi Datta https://www.youtube.com/watch?v=pB0FMshudqE
  3. 3. • DNA:A polymer of deoxyribonucleotides • Self-replicating • Encodes genetic information in all living organisms and some viruses • Found in chromosomes, mitochondria, chloroplast in case of eukaryotes and in nucleoid in case of prokaryotes • Gene: DNA segments that encodes a functional protein • Non-coding regions of DNA have structural and regulatory roles © Dr. Riddhi Datta Deoxyribo nucleic acid (DNA)
  4. 4. Nucleotides and Nucleosides • Nucleotides are the building blocks of nucleic acids. • Nucleotides have three characteristic components: – a nitrogenous base – a pentose – a phosphate • The molecule without the phosphate group is called a nucleoside. • The nitrogenous bases are derivatives of two parent compounds- pyrimidine and purine. © Dr. Riddhi Datta
  5. 5. © Dr. Riddhi Datta Nucleotides and Nucleosides
  6. 6. © Dr. Riddhi Datta Nucleotides and Nucleosides • Ester bond: – Between phosphate and sugar • Glycosidic bond: – Between sugar and base
  7. 7. © Dr. Riddhi Datta Nitrogenous bases
  8. 8. Base Nucleoside Nucleotide Nucleic acid Purines Adenine Adenosine Adenylate RNA Deoxyadenosine Deoxyadenylate DNA Guanine Guanosine Guanylate RNA Deoxyguanosine Deoxyguanylate DNA Pyrimidines Cytosine Cytidine Cytidylate RNA Deoxycytidine Deoxycytidylate DNA Thymine Thymidine (deoxy) Thymidylate (deoxy) DNA Uracil Uridine Uridylate RNA © Dr. Riddhi Datta Nomenclature of nucleic acid components
  9. 9. • DNA consists of two long polymers of deoxyribonucleotides. • The backbone is formed by deoxyribose (sugar) and phosphate groups linked by phosphodiester bonds. • Attached to each sugar is one of the four nitrogeneous bases- adenine, guanine, thymine and cytosine. • Traditionally, DNA is drawn from 5’ to 3’ direction. © Dr. Riddhi Datta Primary structure of DNA 5’ 3’
  10. 10. • DNA is a double helical structure. • The two strands wound around the same axis. • The two stands are anti-parallel, i.e. their 5’ 3’ phosphodiester bonds run in opposite directions. • The two strands are complementary to each other. • The sugar-phosphate backbone forms the periphery while the bases are stacked in the core. © Dr. Riddhi Datta Secondary structure of DNA
  11. 11. • The two strands are held together by hydrogen bonds between two nucleotides in the opposite strands. • Adenosine forms two hydrogen bonds with thymidine. • Guanosine forms three hydrogen bonds with cytidine. • The bases are stacked inside the helix at right angles to the helix axis. • The hydrophobic interaction and van der Waals force between stacked bases are also important to maintain the double helical structure. © Dr. Riddhi Datta Secondary structure of DNA
  12. 12. © Dr. Riddhi Datta
  13. 13. • As the DNA strands wind around each other they leave gaps between each set of phosphate backbone. These grooves are of two types: – Major groove (22 Å wide) – Minor groove (12 Å wide) • The edges of the bases are more accessible in the major groove where proteins (like transcription factors) can interact. © Dr. Riddhi Datta Secondary structure of DNA
  14. 14. • The angle at which two sugars protrude from the base pair (i.e. angle between the glycosidic bond) is around 120°. • As the bases stack on each other, the narrow angle between the sugars on one edge of the bases generates minor groove while the large angle on the other edge generates major groove. • Edges of each base is exposed at the major and minor groove, creating a pattern of hydrogen bond acceptors and donors and of van der Waals surface that identifies the base pair. • These patterns help proteins to specifically recognize DNA sequence without unwinding the double helix. © Dr. Riddhi Datta Secondary structure of DNA
  15. 15. • COVALENT BONDS: – Ester bond (phosphodiester bond): • Between phosphate and sugar – Glycosidic bond: • Between sugar and base • HYDROGEN BOND: – Between the base pairs © Dr. Riddhi Datta Types of bonds present in DNA Bonds affecting DNA stability • HYDROGEN BOND: stabilize – Relatively weak but additive and facilitates base stacking – Hydrogen bond also forms due to formation of a water spine in minor groove • HYDROPHOBIC INTERACTION: stabilize – Hydrophilic groups are oriented outside while hydrophobic groups are outside • STACKING INTERACTION: stabilize – Relatively weak but additive and van derWaals force – The π-π between bases in the helical stacks greatly stabilize the double helix • ELECTROSTATIC INTERACTION: destabilize – Contributed primarily by phosphate groups – Affects intra- and inter-strand interactions – This repulsive forces can be neutralized by positively charged proteins and Na+ ions VAN DER WAALS INTERACTIONS (not within DNA structure) Between major and minor grooves and interacting proteins
  16. 16. UV radiation: • DNA absorbs UV rays at 260 nm. When DNA is denatured from double stranded to single stranded form, its UV absorbance increases cooperatively. • The absorption pattern also increases with increase in GC content. • This is called hyperchromatic shift. • For RNA, increase in absorbance pattern is gradual and irregular. • DNA absorbs UV due to the aromatic bases. © Dr. Riddhi Datta Properties of DNA
  17. 17. UV radiation: • The A260 value of same amount of DNA (e.g 50μg) will differ for single stranded and double stranded forms. – Double stranded DNA:A260 = 1.00 – Single stranded DNA:A260 = 1.37 – Free bases:A260 = 1.60 • Purity of nucleic acids can be estimated by A260 / A280 values: – Pure DNA:A260 / A280 = 1.80 – Pure RNA:A260 / A280 = 2.00 © Dr. Riddhi Datta Properties of DNA
  18. 18. Denaturation: • If double stranded DNA is treated with strong alkali or high temperature, the two strands unwinds and separates.This is known as denaturation. • The temperature at which the two strands separates is called the melting temperature (Tm) and the process is called melting of DNA. Renaturation: • The process by which two separated DNA strands form a double helix again is called renaturation. • This is done by lowering the temperature to 25°C or below. • R.T. Britten and D.E. Kohne (1970) obtained renatured DNA by lowering temperature and determined its C0t value. • C0=primary density; t=interval of time in seconds © Dr. Riddhi Datta Properties of DNA
  19. 19. C0t curve analysis: • It is a technique to determine complexity of genome (DNA). • The technique is based on the principles of DNA reassociation kinetics, • It measures how much repetitive DNA is present in a DNA sample • The technique was developed by R.T. Britten and D.E. Kohne (1970) • Principle: – The rate of DNA renaturation is directly proportional to the number of times the sequences are present in the genome. – Given enough time, all denatured DNA will eventually renature. – The more the repetitive sequence, the lesser the time required for renaturation. – Renaturation also depends on DNA concentration, reassociation temperature,cation concentration and viscosity. • C0t=DNA conc. (moles/L) x renaturation time (s) x buffer factor (constant for buffer composition and accounts for the effect of cations) • Low C0t value: more repetitive sequences; High C0t value: more unique and less repetitive sequences © Dr. Riddhi Datta Properties of DNA
  20. 20. C0t curve analysis: • Application: – Used for determining genomic complexity in different organisms – It can detect the repetitive DNA sequence present in the sample that dominates eukaryotic genomes – This allows DNA sequencing to concentrate on the parts of the genome that are most informative and interesting, which will speed up the discovery of new genes and make the process more efficient. © Dr. Riddhi Datta Properties of DNA
  21. 21. C0t curve analysis: • Example: – Bacterial genome: 99.7% Single copy – Calf genome: 17% High repeat, 23% Intermediate repeat, 60% Single copy © Dr. Riddhi Datta Properties of DNA Pink: High repeat Brown: Intermediate repeat Blue: Single copy
  22. 22. Buoyant density: • DNA has a buoyant density similar to CsCl. Hence DNA can be mixed with CsCl and separated by centrifugation. DNA is acidic: • Though nitrogen bases are present in DNA, it is called nucleic acid. This is because these bases are stacked inside the double helix and are not available for interactions. The backbone is formed by sugar-phosphate where the phosphate is negatively charged and has acidic hydroxyl groups.This makes DNA acidic. • At high ionic concentration, cations shield the negative charge and stabilize the DNA double helix. Ionic induction: • As DNA is acidic, it is negatively charged. During agarose gel electrophoresis it moves towards anode (+). By this process DNA can be separated according to their weight. G:C bond: • G:C bonds are more stable as they form 3 H-bonds and more favorable stacking interactions. So, higher the GC content, more stable is the DNA molecule. Enzymatic hydrolysis: • DNA can be hydrolysed by enzymes called DNases. Enzyme that can hydrolyse DNA sequentially from ends is called exonuclease (Ex: Exonuclease I). Enzyme that can hydrolyse phosphodiester bonds in DNA internally is called endonuclease (Ex: DNase I). © Dr. Riddhi Datta Properties of DNA
  23. 23. • Several structural variants of DNA are possible: – B-DNA (most prevalent conformation in physiological conditions) – A-DNA – Z-DNA © Dr. Riddhi Datta Conformations of DNA
  24. 24. Z-DNA: • Z-DNA is a left handed double helical structure with a zig-zag pattern. • Biologically active but transient structure induced by biological activity. • The major and minor grooves show little difference in width. • Conditions for Z-DNA: – Alternating purine-pyrimidine – Negative DNA super coiling – Low salt and cation concentration – Physiological temperature 37 °C and pH 7.3 – 7.4 • Significance: – Provides torsional strain relief (supercoiling) while DNA transcription occurs. – Potentiality to form Z-DNA also correlates with regions of active transcription © Dr. Riddhi Datta Conformations of DNA
  25. 25. A-DNA: • A-DNA is a right handed double helical structure fairly similar to B- DNA. • Biologically active but transient structure induced by biological activity. • The structure is shorter and more compact. • Major grooves are deeper and minor grooves shallower than B-DNA. • Conditions for Z-DNA: – Dehydrated samples of DNA (ex: samples for crystallographic studies) – Found in DNA-RNA hybrids and double stranded RNA. © Dr. Riddhi Datta Conformations of DNA
  26. 26. Geometry attributes A-DNA B-DNA Z-DNA Helix sense Right-handed Right-handed Left-handed Repeating units 1 base pair (bp) 1 bp 2 bp Rotation/bp +33.6° +36° -30° Mean bp/turn 11 10 12 Inclination of bp to axis +19° -1.2° -9° Rise/bp along axis 2.3Å 3.4Å 3.8Å Rise/turn of helix 24.6Å 34Å 45.6Å Mean propeller twist +18° +16° 0° Glycosyl angle anti anti Pyrimidine: anti; Purine: syn Sugar pucker C3’ endo C2’ endo C: C2’ endo; G: C2’ exo Helix diameter 26Å 20Å 18Å © Dr. Riddhi Datta Conformations of DNA B-DNA
  27. 27. © Dr. Riddhi Datta anti and syn glycosyl angle The bond joining the 1′-carbon of the deoxyribose sugar to the heterocyclic base is the N-glycosidic bond. Rotation about this bond gives rise to syn and anti conformations. Rotation about this bond is restricted and the anti conformation is generally favored, partly on steric grounds.
  28. 28. • The bases in a base pair are usually not coplanar; they instead are twisted about the hydrogen bonds that connect them, like the blades of a propeller. • The dihedral angle that defines the non-coplanarity is called the propellor twist angle. © Dr. Riddhi Datta Propeller twist
  29. 29. • The pentose sugar ring in DNA is not planar.This non-planarity is called sugar puckering. • Sugar puckering can influence both nucleotide incorporation and extension by polymerases. • RNA polymerases preferentially incorporate nucleotides having C3’-endo pucker while DNA polymerases prefer C2’-endo pucker. • If the puckered C atom is displaced towards the C5 atom, then that twisted form is called endo. If it is displaced away from the C5 atom, the form is called exo. • For example, if C3 is twisted towards the C5, it is called C3’-endo. © Dr. Riddhi Datta Sugar pucker
  30. 30. © Dr. Riddhi Datta NonWatson-Crick base pairing Watson-Crick base pairing: • Planar base-pairing • A::T and G:::C • Dimensions of both base pairs are similar, i.e. C1-C1 distances are nearly equal • β-glycosidic bonds attached to same edge of the base pair which defines major and minor grooves • There is potential for further H-bonding which is important for sequence specific protein binding Watson-Crick base pairing: • A-T pairs use N1 of adenine as H- bond acceptor. This geomerty is more stable for double helices. Hoogsteen base pairing: • A-T pairs use N7 of adenine as H- bond acceptor. Seen in crystals of monomeric A-T base pairs. • Co-crystallized monomeric G-C pairs always follow Watson-Crick base pairing.
  31. 31. © Dr. Riddhi Datta

×