SU 3

• Primary structure


  Nucleotide sequence
• Primary structure


  Determines higher order structure
• Nucleotide sequence is the physical representation of genetic information in organisms
• Gregor Mendel discovers the laws of inheritance


  1865
• Human genome sequenced


  200s
• After Avery et al demonstrated DNA to be the genetic material, others further investigated the structure of DNA

  
• Chargaff evaluated base composition of different DNAs

  1. Four bases found in DNA

  2. Bases do not occur in equimolar amounts

  3. Amount of each varies between species

  4. Base pairs found in 1:1 ratio A:T, C:G

  5. [pyrimidine]=[purines]

  
• Rosalind Franklin develops X-ray picture of DNA
• DNA in isolated form

  • 2 strands
  • Long and threadlike
  • Takes on a helical form
  • Strands are complementary and run in opposite directions (antiparallel)
  • Strands held together by bonding interactions between unique base pairs
  • Each base pair: purine – pyrimidine
  • Base pairing is very specific (A:T, G:C)
  • Specificity in bp → spatially equivalent units (backbone-to-backbone distance A:T = 1.11 vs G:C 1.08nm)

  
• Base pairing not only conforms to Chargaff's rules/W + C rules, it also contains hereditary info: sequences of bases in 1 strand is complementary to sequence of bases in the other strand
• When strands separate and replicate, base pairing will specify the nucleotide sequence of the new strand and ensure the daughter molecules produced will be identical to parent

  
• Due to double stranded helical nature, size of DNA is represented as #base pairs
• DNA is long, compacted into chromosomes to fit the nucleus of our cells

  
• Prokaryotic chromosomes


  • Circular
• Eukaryotic chromosomes


  • Linear
  • DNA winds around histones to form nucleosomes
  • Electrostatic interaction between histones and anionic P groups in the DNA backbone
  • Non histone chromosomal proteins regulate which genes are transcribed at a specific time

  
• DNA uses 4 digits (A,C,G,T) to encode biological info
• Determining the primary structure of DNA

  • Fewer sites for selective cleavage
  • Distinct sequences difficult to recognize
  • Likelihood of ambiguity is big
• Tools used by scientists to determine structure of DNA/order of nucleotides

  1. Restriction enzymes (cleave DNA at specific sites → fragments of manageable size)

  2. Electrophoresis (method to separate fragments based on size)

  
• Chain termination method for nucleotide sequencing

  1. 2 strands separate

  2. Each strand now serves as a template strand

  3. Template strand incubated with 4dNTPs

  4. Enzymes: DNA polymerase

  5. Nucleotide sequence of 1 strand copied in complementary fashion to new strand → 2 daughter strands

  6. Invitro: reaction will take place provided primer (oligonucleotide capable of forming a short stretch of dsDNA when it bp ssDNA)

  7. Primer important: free 3'OH to which growing polypeptide is added

  8. Chain grows in 5' to 3' direction

  
• Sanger sequencing/chain termination method

  • Traditional protocol for sequencing
  • A template DNA (the single-stranded DNA to be sequenced)
  • With a complementary primer annealed at its 3'-end
  • Template is copied by DNA polymerase in the presence of four deoxynucleotide substrates (dATP, dCTP, dGTP, dTTP)
  • Small amounts of the four dideoxynucleotide analogs of these substrates, each of which carries a distinctive fluorescence tag
  • Occasional incorporation of dideoxynulceotide terminates further synthesis of that complementary strand
  • The nested set of terminated strands can be separated by capillary electrophoresis and identified by laser fluorescence spectroscopy
  • New strands of varying length produced
  • Each new syntheiszed strand: dideoxynucleotide at its 3' end
  • Each dideoxynulceotide is unique, has unique FL tag
  • FL tag color reveals which base (as specified by the template strand) is incorporated

  
• Reading the dideoxy sequencing gels

  1. Products are visualized by fluorescence spectroscopy

  2. Separate based on size

  3. Smallest fragments migrate fastest – are at the bottom of the gel

  4. Sequence of nucleotides in newly synthesized strand given by the order of FL colors

  
• Several adaptions to this method has led to next generation and ultra high throughput sequencing methods
• Next generation sequencing methods

  Sequencing still via sequencing by synthesis where: DNA polymerase catalyzes synthesis of complementary DNA strand, Machine digitally records addition of a base, Digital form stores quantitative data, DNA pulled through nanospores, Measure change in electrical conductance of nanospore, Readout of electrical changes ~ respective nucleotide, Pyrophosphate release coupled to light emission, Ion torrent measures: pH change due to proton release when deoxynucleotide added

  
• DNA structure


  • Polynucleotide strands are flexible
  • 6 degrees of freedom in the sugar-phosphate backbone due to 6 single bonds per segment along the chain
  • 7th degree of freedom at the glycosidic bond
  • Sugars are also not flat but puckered (folded/tilted)
  • Converts the DNA ladder to a helix

  
• DNA double helix


  • DNA comprises 2 strands
  • H2 bonds between bases
  • H2 bonds possible when strands antiparallel
  • Polar sugar-phosphate backbone oriented outward
  • Bases stacked inside and are hydrophobic
  • Distance between bases at 0.6 nm (distance between sugars along a strand)
  • Water can fit between hydrophobic surfaces of bases so this conformation is energetically favourable
  • Ladder converts to helical conformation by twisting right
  • Base pair rungs/distance are now closer 0.34nm
  • No effect on sugar-sugar distance
  • Helix repeats every 10bp thus 0.34nmx10bp = 3.4nm pitch
  • This is the major conformation of DNA in solution (B-DNA)

  
• Watson-Crick base pairs


  • Have identical dimensions
  • DNA helix is stabilised by H-bonds (H2 bonds between base pairs adds less stability, When strands separate the H2 bonds between bases replaced by H2 bonds between bases and surrounding H2O, Polar atoms in sugar phosphate backbone also hydrogen bonds with H2O)
  • Electrostatic interactions (Negatively charged sugar phosphate backbone repel each other so sugar P backbones are kept apart and base pairing allowed, Negatively charged phosphates are exterior to the helix so repulsive effects minimized, Negative charges shielded by cations)
  • Van der Waals interactions (Core of helix = base pairing, Base pairs stack via electronic interactions and hydrophobic forces, Due to the way bp occurs, sugars within the nucleotides have opposite orientations, Thus the 2 strands are antiparallel/running in opposite orientations, Glycosidic bonds of each bp not directly opposite each other – common diameter, Sugar phosphate not equally spaced along the helix axis, Grooves different sizes)
  • Hydrophobic interactions

  
• 1. D DNA

  • Phosphate backbone is offset centre of the helix
  • Grooves between the 2 strands are of different sizes (Major grooves - more open, exposes nucleotide bp, Minor grooves - more constricted, blocked by the deoxyribosyl units linking the bp)
  • Protein binding occurs at the major groove
  • Protein binding specific for the nucleotide sequence of DNA

  
• Conformations of DNA


  • B form - DNA stable but has inherent flexibility in sugar phosphate backbone, glycosidic bond and right hand twist, Base pairing remains the same, Base pair rotations are another conformation variation (Helical – bp rotate with respect to each other, Propellor twist – rotation around a different axis)
  • A-right-handed (Base pairs are around rather than centred on helical axis, Distance to complete 1 helical turn/PITCH is 2.46nm, 21 turn requires 11bp to complete, Bp tilted 19 degrees to axis, Bp every 0.23nm along axis, Shorter and squat, Polypurine residues, Dehydrated environments, Double stranded regions of RNA = A conformation)
  • Z-left-hand conformation of B-DNA (Alternating pyrimidine:purine sequence, N-glysosyl bond of G residues flips 180 degrees, Sugar phosphate back bone aligned to a zigzag course with left hand orientation, More elongated and slimmer than B-DNA, Occurs under conditions of high ionic concentrations)
  • Methylation favours B to Z conformation, Methylation has role in gene regulation, Z form may have affect gene expression

  
• DNA characteristics


  • DNA is flexible, Structure can be disoriented/deformed, Elastic motion (bases and phosphate backbone), Slight bending of helix, Proteins recognizes DNA sequences, influencing gene expression

  
• Intercalating agents can distort double helix structure

  1. Aromatic, hydrophobic molecules can slip between bases

  2. Bases move apart

  3. Unwinding of helix (makes room for aromatic compounds)

  4. Favourable van der Waals forces between aromatics and bases versus between bases themselves
• Denaturing DNA by temperature

  1. pH, temperature, ionic strength can disrupt bp

  2. When bp is disrupted, strands no longer together

  3. Helix is denatured

  4. If temperature = denaturing agent, helix is said to melt

  5. Separation of the strands followed spectrophotometrically

  6. Interactions between bp via electron clouds

  7. There is a subsequent increase in absorbance at 260nm because unstacking of pb alleviates suppression of UV absorption = hyperchromic shift

  8. Midpoint of increase in absorbance = melting temperature

  9. DNAs differ in their melting temperature values because of differences in G:C content

  10. Higher GC content/ionic strength the higher the Tm (>bp stacking energies)

  11. Melting dependent on ionic strength, Lower ionic strength, lower melting temperature

  12. Cations supress – verily charged phosphate group charges
• Extreme pH denatures DNA

  1. At pH 10, DNA base are deprotonated, bp is limited → denaturing

  2. At pH 2.3 excessive protonation
• Denaturation


  Disruption of base pairing in DNA, causing the strands to separate

  
• Factors that can denature DNA

  • Temperature
  • pH
  • Ionic strength
• DNA denaturation


  1. Disruption of base pairing

  2. Separation of strands

  3. Helix is denatured

  4. Strands melt apart

  
• Hyperchromic shift


  Increase in absorbance at 260nm due to unstacking of base pairs when DNA strands separate

  
• Melting temperature (Tm)


  Midpoint of increase in absorbance, indicates DNA stability
• DNA with higher GC content

  Has higher melting temperature
• Lower ionic strength


  Lowers DNA melting temperature
• Renaturation


  Realignment of complementary DNA strands to reform base pairs