Differentiation between exonucleases and endonucleases and between haplotomic and diplotomic endonucleases using 3-h-dna-coated wells of plastic depression plates as substrate.

Using our new method for assaying DNases with radioactively labeled DNA bound to wells of plastic depression plates as substrate, we could distinguish between endonucleases and exonucleases and between haplotomic and diplotomic endonucleases. Oligonucleotides smaller than 30 detach from the DNA binding sites of the well into the reaction mixture. Thus, a lag period was evident before endonucleases produced small soluble oligonucleotides, while exonucleases released mononucleotides or short oligonucleotides without any lag period. Haplotomic and diplotomic endonucleases were detected because of the different rates in which they produce small soluble oligonucleotides which were expressed in different lag periods. Under conditions in which the haplotomic DNase 1 changes its mode of action to become a diplotomic enzyme, the shift was clearly detected by a change in the lag period in the well assay.