VPB 112: General Veterinary Biochemistry (1+1)

DNA DENATURATION

• Complete unwinding and separation of the complementary single strand is called denaturation. Denatured DNA consists of two single strands. Double stranded DNA is stable than the separated strands.
• The strength of double strand DNA depends on ionic strength, its pH and base compositions. The double strand of DNA can be denatured by raising pH or temperature or drastically lowering the ionic strength.When a DNA solution is heated enough, the non-covalent forces that hold the two strands together weaken and finally break.When this happens, the two strands come apart in a process known as DNA denaturation, or DNA melting.The temperature at which the DNA strands are half denatured is called the melting temperature, or Tm. The amount of strand separation, or melting, is measured by the absorbance of the DNA solution at 260nm.When the two strands separate the absorbance rises 30% - 40%.This is called hyperchromic shift.
  
• When the denatured DNA is brought to physiological condition, the single strands come back together to regenerate the B form of DNA.
• Due to the –ve charge of phosphate groups, the two strands of DNA repel one another. When salt is added, The cations (monovalent or divalent) such as Na⁺, Mg²⁺,Mn²⁺ and Co²⁺ interact with the ionic phosphate groups of DNA and act as a shielding agent( repulsive force is reduced), hence the DNA is stabilized.
• Enzymes that catalyzes the hydrolysis of phosphodiester in nucleic acids are called nucleases. Ribonuclease acts on RNA and deoxyribonuclease acts on DNA. Nuclease can be further classified as exonuclease, which catalyzes the hydrolysis of phosphodiester linkage to release nucleotide residues from only one end of polynucleotide chain, or endonuclease, which catalyzes the hydrolysis of polynucleotide linkage at various sites within the polynucleotide chain.
• At high temperature the phosphatediester bond may be broken into pieces but in the presence of alkali the DNA is unwound but not broken.
• Presence of formaldehyde causes irreversible denaturation.
• RNA is degraded under alkaline solution, whereas DNA is stable.
  Forms of DNA
• There are at least 6 forms of DNA. A, B, C, D, E and Z of which three are the major structural forms of DNA - the A, B and Z. The predominant form of DNA is the ‘B’ form, which is a right-handed helix with 10 base pairs per turn of the helix. This form is present in all the cells and its structure has been described above.
• The ‘A’ form is produced by dehydrating the ‘B’ form. It is also a right-handed helix. 11 base pairs are present per turn.
• The ‘Z’ form is a left-handed helix with 12 base pairs per turn. Due to its zigzag structure it is called as ‘Z’ form.

Characteristics	A-DNA	B-DNA	C-DNA	Z-DNA
Conditions	75% relative humidity; Na+, K+, Cs+ions	92% relative humidity; Low ion strength	60% relative humidity; Li+ ions	Very high salt concentration
Shape	Broadest	Intermediate	Narrow	Narrowest
Helix sense	Right -handed	Right-handed	Right-handed	Left-handed
Helix diameter	25.5A◦	23.7A◦	19.0A◦	18.4A◦
Rise per base pair (‘h’)	2.3A◦	3.4A◦	3.32A◦	3.8A◦
Base pairs per turn of helix (‘n’)	11	10.4	9.33	12 (=6 dimers)
Helix pitch	25.30A◦	35.36A◦	30.97A◦	45.60A◦
Rotation per base pair	+32.72A◦	+34.61A◦	+38.58A◦	-60◦(per dimer)
Base pair tilt	19◦	1◦	7.8◦	9◦
Glycosidic bond	anti	anti	-	anti for C, T & Syn for A, G
Major groove	Narrow and very deep	Wide and quite deep	-	Flat
Minor groove	Very broad and shallow	Narrow and quite deep	-	Very narrow and deep