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Nucleic Acids Research, 1989, Vol. 17, No. 10 3735-3745
© 1989

ENZYMOLOGY

Selective hydrolysis by exo- and endonucleases of phosphodiester bonds adjacent to an apurinic site

Michael Weinfeld*, Michel Liuzzi and Malcolm C. Paterson

Laboratory of Molecular Genetics and Carcinogenesis, Department of Medicine, Cross Cancer Institute Edmonton. Alberta T6G 1Z2, Canada

*To whom correspondence should be addressed.

Received January 31, 1989. Revised April 14, 1989. Accepted April 14, 1989.

Partial depurination of d-ApA produced two UV260nm-absorbing isomers, d-SpA and d-ApS (where S represents the depurinated deoxyribose sugar), that provided simple model compounds with which to examine, by HPLC, the response of nucleases to phosphodiester bonds flanked 3' or 5' by an apurinic site. The structural identity of each compound was established by (i) reaction with methoxyamine to confirm the presence of an abasic deoxyribose group, and (ii) degradation of d-SpA under mild alkaline conditions to distinguish it from d-ApS. At an enzyme concentration which led to complete hydrolysis of d-ApA, snake venom phosphodiesterase readily cleaved d-SpA to 5'-dAMP but had no discernible effect on d-ApS. Calf spleen phosphodiesterase also failed to act on one isomer, in this instance d-SpA, but additionally reacted at a much slower rate (–100 fold) with d-ApS than with d-ApA. Three single-strand specific endonucleases, nuclease PI, nuclease SI and mung bean nuclease, all responded in an identical manner, hydrolysing d-ApS but not d-SpA. The possibility that the aldehyde group at the AP sites might be responsible for some of these observations was rejected after repeating the enzyme digestions with the methoxyamine-capped molecules and observing no differences from the reactions with d-SpA and d-ApS.


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