DNA stability: Difference between revisions
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(New page: 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 f...) |
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==Depurination== | ==Depurination== | ||
* Depurination involves the loss of purine bases forming abasic sites | * 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) | * 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 | * DNA under physiological conditions has been estimated to depurinate at a rate of <math>3\cdot 10^{-11}</math>/sec at 37C and pH 7.4 <cite>lindahl72</cite> | ||
* 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== | ==Deamination== | ||
* Cytosine can be spontaneously deaminated to form uracil. | * Cytosine can be spontaneously deaminated to form uracil. | ||
* Cytosine in native DNA is estimated to deanimate with a rate constant of <math>7\cdot 10^{-13}</math>/sec at 37C and <math>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>(8-40)\cdot 10^{-10}</math>/sec at 60C and about <math>(4-13)\cdot 10^{-10}</math>/sec at 37C | |||
==Strand cleavage== | ==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 <cite>lindahl72b</cite> | |||
* 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 | * 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 | * 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== | ==References== | ||
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#frederico93 pmid=8329382 | #frederico93 pmid=8329382 | ||
#lindahl72 pmid=4626532 | #lindahl72 pmid=4626532 | ||
#lindahl72b pmid=4559796 | |||
</biblio> | </biblio> | ||
[[Category:DNA]] |
Latest revision as of 12:31, 27 April 2007
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
- Lindahl T and Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3610-8. DOI:10.1021/bi00769a018 |
- Lindahl T and Andersson A. Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3618-23. DOI:10.1021/bi00769a019 |
- Frederico LA, Kunkel TA, and Shaw BR. Cytosine deamination in mismatched base pairs. Biochemistry. 1993 Jul 6;32(26):6523-30. DOI:10.1021/bi00077a005 |