I am wondering how DNA glycosylase works from the chemical point of view. Sometimes in papers I can see that they are doing activity assays with 1M NaOH and I am trying to understand why using 1M NaOH is more factual? Thank you for any help.


1 Answer 1


DNA glycosylases bind to dsDNA, flip a base out of the base-paired double-helical structure and cleave it off, leaving the sugar-phosphate backbone intact. This base removal renders the DNA susceptible to alkaline cleavage.

In this recent paper:

Miyazono et al. (2014) A sequence-specific DNA glycosylase mediates restriction-modification in Pyrococcus abyssi. Nature Communications 5: #3178

the authors monitor the activity of a DNA glycosylase using this phenomenon: after treating an oligonucleotide with increasing amounts of the enzyme R.PabI they treat the DNA with NaOH so that the oligo is cleaved, allowing detection of the result of enzyme action on a polyacrylamide gel, as you can see from this image, taken from their Figure 3.

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Incidentally the same phenomenon is exploited when DNA is transferred to a membrane for Southern blotting. After electrophoresis the gel is soaked in acid which causes depurination (analogous to DNA glycosylase). It is then transferred to an alkaline solution. Although protocols often refer to this as a neutralisation step it actually breaks the DNA at the apurinic sites and separates the strands. This cleavage is important for effective transfer of larger fragments from the gel to the membrane.


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