Nucleic Acids

1. 5' 5' 3' 3' Nucleic Acids • Nucleic acid: are polymers of Nucleotides linked with 3’, 5’- phosphodiester bonds • Nucleotide residues are all oriented in the same direction (5’ to 3’) giving the polymer directionality. • The sequence of DNA molecules is always read in the 5’ to 3’ direction

2. 5' 3' 5' 3' 3' Phospho diester bond formation 5' 5' 3'

3. 5' 5' 3' 3' • Nucleotide monomers are joined by 3’-5’ phosphodiester linkages to form nucleic acid (polynucleotide) polymers

4. 5' 3' • Phosphodiester bonds can be cleaved hydrolytically by chemicals or enzymatically by deoxyribonucleases (DNA) or ribonucleases (RNA)

5. DNA • 1o Structure - Linear array of nucleotides and their sequence can be determined by different methods. • 2o Structure – double helix • 3o Structure - Super-coiling, stem-loop formation • 4o Structure – Packaging into chromatin DNA Secondary structure • DNA is double stranded with antiparallel strands • Right hand double helix • Three different helical forms (A, B and Z DNA.

6. Properties of DNA Double Helix * The two chains are coiled around a common axis * The chains are paired in an anti-parallel manner * Distance between the 2 sugar-phosphate backbones is always the same, give DNA molecule a regular shape. * Plane of bases are oriented perpendicular to backbone * Hydrophilic sugar phosphate backbone winds around outside of helix * Noncovalent interactions between upper and lower surfaces of base-pairs (stacking) forms a closely packed hydrophobic interior. * Hydrophobic environment makes H-bonding between bases stronger (no competition with water)

7. Bases from two adjacent DNA strands can hydrogen bond • Adenine pairs with thymine using two H-bonds • Guanine pairs with cytosine using three H-bonds

8. 5' 3' 3' 5' H-bonding of adjacent antiparallel DNA strands form double helix structure

9. Hydrophobic Interior with base pair stacking Sugar-phosphate backbone View down the Double Helix

10. Structure of DNA Double Helix • Right handed helix • Rise = 0.33nm nm/nucleotide • Pitch = 3.4 nm / turn • 10.4 nucleotides per turn • Two groves – major and minor

11. * Within groves, functional groups on the edge of base pairs exposed to exterior * involved in interaction with proteins. Factors stabilizing DNA double Helix * Hydrophobic interactions – burying hydrophobic purine and pyrimidine rings in interior * Stacking interactions – van der Waals interactions between stacked bases. * Hydrogen Bonding – H-bonding between bases * Charge-Charge Interactions – Electrostatic repulsions of negatively charged phosphate groups are minimized by interaction with cations (e.g. Mg2+)

12. Three major structural forms of DNA A: right-handed, short and broad, 2.3 A, 11 bp per turn B: right-handed, longer, thinner, 3.32 A, 10 bp per turn Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn, Found in G:C-rich regions of DNA

13. Structural forms of the double helix • Three major structural forms of DNA • B-form, described by Watson and Crick • It is right handed helix with 10 residues per 360° turn of the helix • The plane of bases perpendicular to helical axis • Chromosomal DNA consists primarily of B-DNA • A-DNA form • Is produced by moderately dehydrating the B form • It is right-handed helix • Contains 11 base pairs per turn • The planes of the base pairs are tilted 20 ° away from the perpendicular to helical axis • Z-DNA form • contains 12 bp per turn • Is left handed helix • Contains 12 about 12 base pair per turn • The deoxyribose phosphate backbone forms a “Zigzag structure”

14. Right handed helix

15. Structure of DNA Double Helix

16. Nucleoid -Prokaryotic organism contains a single, double stranded, supercoiled, circular chromosome, and it is associated with histone-like proteins and some RNA to form a nucleoid Plasmid • Most species of bacteria contain small, circular, extrachromosomal DNA molecules that called Plasmid. • Plasmid DNA carries genetic information and undergoes replication that may or may not related to the chromosomal division. • Plasmid carries genes that convey antibiotic resistance to the host bacterium and may facilitate the transfer of genetic information from one bacterium to another • Plasmids used as vectors in recombinant DNA technology

17. Separation of the two DNA strands in the double helix Heating up to 70 – 90C° the DNA double helix denatures, H-bonds are broken, bases unstack, and the strands separate. Renaturation (annealing)at lower temperatures occurs in 2 steps. 1. Complementary bases pair. 2. The rest of the structure forms cooperatively; it “zips-up”. DNA with high G:C content denatures at a higher Temp. than A:T rich segments.

18. DNA sequence Determines Melting Point • Double Strand DNA can be denatured by heat (get strand separation) • Can determine degree of denturation by measuring absorbance at 260 nm. • Conjugated double bonds in bases absorb light at 260 nm. • Base stacking causes less absorbance. • Increased single strandedness causes increase in absorbance

19. Melting Point • Single strand has higher relative absorbance at 260 than dose double stranded DNA. • Increasing the denaturation  increases the absorbance Melting point: the temperature at which the half of the helical structure is lost

20. DNA sequence Determines Melting Point * Melting temperature related to G:C and A:T content. * 3 H-bonds of G:C pair require higher temperatures to denture than 2 H-bonds of A:T pair.

21. DNA 3o Structure • Supercoiling • Cruciform structures DNA supercoiling: • Supercoiling: means the coiling of the coil. • Typical phone cord is coiled like a DNA helix and the coiled cord can itself coil in a supercoil • A number of measurable properties of supercoiling have been established

22. Significance of DNA Supercoiling: A supercoiled DNA molecule is more compact than a relaxed DNA molecule of the same length supercoiled DNA moves faster than relaxed DNA in centrifuged There are many topological parameters that describe supercoling

24. DNA Supercoiling

25. Certain DNA sequences adopt unusual structures • Palindrome: a word that spelled identically reading forward or backward (ROTATOR). • This term is applied to DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA, such sequences are self-complementary within each strand • When inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat.

26. Palindromes have potential to form hairpin or cruciform

27. Cruciform: cross-shaped structure Can form intrachain base pairing

28. DNA 4o Structure: Chromosome Structure • In chromosomes, DNA is tightly associated with proteins • Human DNA’s total length is ~2 meters! • This must be packaged into a nucleus that is about 5 micrometers in diameter • This represents a compression of more than 100,000! • It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments • Nucleosome Structure • Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins • % major histone proteins: H1, H2A, H2B, H3 and H4 • Histone octamers are major part of the “protein spools” • Nonhistone proteins are regulators of gene expression

30. Nucleosome Structure • High content of Lysine and arginine (+ve charge) • 4 major histone (H2A, H2B, H3, H4) proteins for octomer • 200 base pair long DNA strand winds around the octomer • 146 base pair DNA “spacer separates individual nucleosomes • H1 protein involved in higher-order chromatin structure. • Chromatin looks like beads on string

31. Organization of Eukaryotic DNA

32. Organization of Eukaryotic DNA

33. The End

34. DNA Supercoiling • Supercoiling is a property of circular DNA not linear • The number of helical turns in a linear 260-bp DNA duplex in the B-DNA form is 25 (260/10.4) • Joining the two ends Relaxed circular DNA (also has 25 helical turn)

35. Another circular DNA can be formed by unwinding the linear duplex by two turns before joining its ends This circular DNA contains 23 turns of B-helix and unwound loop In vivo most DNA is negatively supercoiled

36. A supercoiled DNA molecule is more compact than a relaxed DNA molecule of the same length supercoiled DNA moves faster than relaxed DNA in centrifuged Topological parameters that describe supercoling • Linking number Lk: the number of times one strand of DNA winds around the other in the right-handed direction. Molecules that differs only in linking number are called topological isomers or topoisomers • Number of turns of Watson-Crick helix “T” (Twisting number) • Number of turns o superhelix “W” (writhing number) L=T + W T, W can be non-integral but L should be integral Supercoiled DNA is favored over unwound DNA because it contains more paired bases Enzymes called topoisomerases or gyrases can introduce or remove supercoils

38. 4 major histone (H2A, H2B, H3, H4) proteins for octomer • 200 base pair long DNA strand winds around the octomer • 146 base pair DNA “spacer separates individual nucleosomes • H1 protein involved in higher-order chromatin structure. • W/O H1, Chromatin looks like beads on string