DNA stability

Although DNA is generally viewed as a stable molecule, many conditions can cause loss of DNA bases or strand breakage.

Depurination

• Depurination involves the loss of purine bases forming abasic sites
• Depurination is one of the two limiting factors in chemical synthesis of long DNA oligos (the other is coupling efficiency)
• DNA under physiological conditions has been estimated to depurinate at a rate of [math]\displaystyle{ 3\cdot 10^{-11} }[/math]/sec at 37C and pH 7.4 [1]
• Heating DNA for 10m@100 at pH 7.0 leads to about 1 apurinic site per 1000 base pairs
• The activation energy of depurination is around 29 kcal/mol
• Higher temperatures lead to faster depurination
• Denatured DNA depurinates at about 4 times the rate of dsDNA @ pH 7.4
• Methylated As (6-methyladenine) found in bacteria are depurinated 4 times faster than the unmethylated purine bases
• Depurination decreases at higher pH (thus acidic conditions favor depurination)
• Depurination proceeds more rapidly in buffers of low ionic strength
• Depurination is correlated with lower transformation efficiency
• Depurination is independent of sequence

Deamination

• Cytosine can be spontaneously deaminated to form uracil.
• Cytosine in native DNA is estimated to deanimate with a rate constant of [math]\displaystyle{ 7\cdot 10^{-13} }[/math]/sec at 37C and [math]\displaystyle{ 10^{-10} }[/math]/sec at 70C
• Single stranded DNA deaminates significantly faster (> 100 times) than double stranded DNA
• Mispaired Cs (C:C and T:C) are 1-2 orders of magnitude more likely to deaminate than in double stranded DNA (similar to single stranded DNA)
• The rate of deamination of C mispairs is around [math]\displaystyle{ (8-40)\cdot 10^{-10} }[/math]/sec at 60C and about [math]\displaystyle{ (4-13)\cdot 10^{-10} }[/math]/sec at 37C

Strand cleavage

• Under physiological conditions (pH 7.4, ionic strength of 150mM with 10mM Mg++ ions), the lifetime of a phosphodiester bond at an abasic site is 190 hours @ 37C [2]
• Abasic sites are alkali-labile. Under mildly alkaline conditions, β-elimination occurs which nicks 3' to the abasic site leaving a 5'-P on the downstream fragment
• Under strong alkaline conditions, δ-elimination will occur after β-elimination which completely removes the abasic site leaving a 3'-P on the upstream fragment and a 5'-P on the downstream fragment
• Amines (such as amino acids or polyamines like putrescine, spermidiine, and spermine) greatly increase the rate of strand breakage at abasic sites

References

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