Selective hydrolysis by exo- and endonucleases of phosphodiester bonds adjacent to an apurinic site.
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 (approximately 100 fold) with d-ApS than with d-ApA. Three single-strand specific endonucleases, nuclease P1, nuclease S1 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.
Weinfeld
M
M
Department of Medicine, Cross Cancer Institute, Edmonton, Alberta, Canada.
Liuzzi
M
M
Paterson
M C
MC
D001080:Apurinic Acid / Q000138:chemical synthesis / Q000378:metabolism*; D003435:Crotalid Venoms; D004720:Endonucleases / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D006868:Hydrolysis; D007202:Indicators and Reagents; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011119:Polynucleotides / Q000378:metabolism*; D013379:Substrate Specificity
D003435:Crotalid Venoms; D007202:Indicators and Reagents; D011119:Polynucleotides; D001080:Apurinic Acid; D004720:Endonucleases; D005092:Exonucleases; D010727:Phosphoric Diester Hydrolases