The Specificity of a Neutral Deoxyribonuclease from Cancer pagurus

Abstract

The recently isolated neutral deoxyribonuclease from crab (Cancer pagurus) testes has been characterized in its mode of action and its specificity. The enzyme is a typical endonuclease, forming 5'-phosphate oligonucleotides of large average size; after extensive digestion of calf thymus DNA over 75% of the fragments have a size larger than pentanucleotides and mononucleotides are absent. As far as specificity is concerned, thymidine is very abundant in the 5'-penultimate position ( x 50 %) and in the 3'-terminal position (37 %) and almost absent in the 5'-terminal position (z 1 %), the values quoted concerning Escherichiu coli digests of average size (P,,) between 50 and 10.Recent results on the specificity of deoxyribonucleases (DNAases) and on their use in assessing the frequency of the short nucleotide sequences recognized and split by these enzymes (see [l] for a brief review) encouraged us to screen a number of deoxyribonucleases for specificity. In the present work, we have investigated the specificity of a neutral DNAase recently isolated from Cancer pagurus testes [2]. A new procedure of preparation of the enzyme, based on the method originally developed for spleen acid DNAase [3], has been set up. The specificity of the enzyme has been determined using the labelling and separation approaches recently established for the 3'-terminal nucleotides and the 5'-terminal dinucleotides [4,5] of the oligonucleotides formed by the crab DNAase. The 5'-terminal dinucleotides released from Escherichiu coli DNA (51 "/, dG + dC) by the enzyme are very characteristic in that thymidine is almost absent from the terminal position (z 1 %) and predominates on the penultimate position (z 50 %) ; thymidine also is the most frequent (37 %) 3'-terminal nucleotide liberated by the enzyme. IS, 203-208 (1970). MATERIALS AND METHODSThe DNA preparations used in the present work were described elsewhere [6,7].Crab DNAase digestions were carried out in 0.005 M Tris-HC1 pH 7.2, 0.01 M MgC12. DNA concentration and incubation temperature are indicated in the figure legends. Activity units are defined elsewhere [3].The enzyme was inactivated by shaking the incubation mixture with 0.1 volume of chloroform-isoamyl alcohol (24: 1 ; v/v) on a Whirlimixer (Springfield, Mass.). Digests were extensively dialyzed against distilled water, except where otherwise stated. For the determination of 5'-terminal dinucleotides, they were made 0.05 M in ammonium acetate buffer pH 4.6, and dephosphorylated by incubation at 37 "C for 4 h with 0.005 units of acid phosphatase B per AZ6,, unit of digest; acid phosphatase was inactivated by shaking with chloroform -isoamyl alcohol, as indicated above.DEAE-cellulose -urea column chromatography was done as previously described [6], on undialyzed digests showing a hyperchromic shift of 34 %.The methods for the determination of the 3' and 5'-terminal nucleotides and the 5'-terminal dinucleotides have been described elsewhere [4,8,5] along with the enzyme preparations used.The statistical evaluation of data was done...

The Specificity of Five DNAases as Studied by the Analysis of 5′‐Terminal Doublets

“…J. ): E. coli DNAase was prepared by C. Cordonnier and G. Bernardi according to a procedure to be published elsewhere; crab DNAase was obtained as described by Sabeur et al [7]. DNA digestions, evaluations of average sizes of digests, dialysis and dephosphorylation of the digest (when necessary), were done as already described [lo -13,7].…”

“…For each enzyme, the results obtained on digests having different average sizes were not significantly different and could therefore be averaged out (see below). In the case of the crab enzyme, three hydrolysates having an average size of 25 were analyzed; kinetic results obtained on other DNAs, showed, however, that in this case, too, the composition of the 5'-terminal dinucleotides was not dependent upon the size of the digest, at least in the 50-10 P,, range [7].…”

“…Crab DNAase the experimental percentages of the 3'-terminal nucleotides, previously determined [7,8,12,13,15] by those of the 5'-terminal dinucleotides ( Table 1). The results are shown in Fig.…”

“…The calculated, or statistical, 5'-terminal doublets still reflect, however, the real situation. The distribution of the trinucleotides split by the enzymes was therefore calculated by multiplying [ 7 ] 1 Spleen DNAase…”