non-specific cleavage products in an endonuclease assay - (Nov/19/2006 )

hi everyone...

i'm becoming really dependent on this forum.

i recently purified human abasic endonuclease 1. having used it in an assay with a synthetic radiolabeled oligonucleotide containing a single abasic site (to assay for activity of the purified protein) i find that there is at least one, maybe two nonspecific cleavage products INCLUDING the specific cleavage product. the protein should only cleave at a single site on the oligo, so i should only see the radiolabeled substrate and the smaller cleavage product as bands on the gel.

the enzyme needs a divalent metal cation, and my reaction buffer contains either Mg or Mn, with the cleavage activity being significantly higher in the Mn-containing reaction buffer. a previous bacth of the protein that we had cleaves only at a single site on the oligo, so i know that the oligo is still intact and 'in good shape'.

i am at a loss to explain why this is happening. the oligonucleotide mix is only 80% after purification (according to the manufacturer) the whole oligo, with the remaining 205 being fragments of the original oligo missing one, two, three bases, etc. the enzyme is also probably cutting at the abasic sites in the fragmented substrate oligos, but the potential cleavage products of these fragments are almost the same size as the cleavage product of the original oligo...

i also thought perhaps i had contamination of the purified protein, but again, the nonspecific activity is so robust i don't think a contaminant can have that kind of effect. the only other thing i can think of is that the previous batch of protein we used was dialyzed against PBS, and this batch was dialyzed against a buffer containing HEPES, DTT, and EDTA. could that make a difference in activity though?

i've attached a gel picture. any ideas/ suggestions/ comments? thank you kindly, folks.

If you dialyzed against EDTA you many have removed a native cofactor, different from the Mg++ or Mn++ you are adding. I'd try zinc. Is the pattern you are looking for the Mg++ pattern, and the additional bands in the Mn++ lanes the extra cleavages?

Ca ++

Also another common divalent ion. As phage434 mentioned addition of EDTA might have removed a native factor and thus destabalising your enzyme's structure, hence the nonspecific cleavage.

A test to this idea would be to add some EDTA to your previous batch of protein that was shown to be specific and see if the nonspecific cleavage pattern appears.

thanks for your suggestions. i don't have any of the previous batch of protein left, unfortunately. zinc apparently inhibits this protein...as does nickel.