Ssp5230l, a novel isoschizomer ofAatllfromStreptomycesrecognizing 5′-GACGT/C- 3′

We have isolated Ssp5230I, a novel class-II restriction endonuclease from Streptomyces species recognizing the palindromic sequence 5'-GACGT/C-3' generating 3'-protruding ACGT-tetranucleotides. With respect to its isoschizomer Aatll it can be isolated in higher purity and stability. A comparison of cleavage patterns obtained with Ssp5230I using lambda, Ad-2, phiX174, pBR328, pBR322 and pUC18 DNAs of known nucleotide sequence (Figure 1, lanes 3-8) with computer-derived mapping data (1) predicts the sequence 5'-GACGTC-3'. The recognition sequence was confirmed by parallel digestion of lambda DNA with its isoschizomer Aatll (2) (Figure 1, lane 2) resulting in both cases in fragments of approximately 14000, 12000, 5100, 4300, 3700, 3300, 2900, 1800, 1100 and 300 bp which correlate with the computer-derived length of 14062, 11770, 5109, 4289, 3731, 3316, 2906, 1849, 1134, 307 and 29 bp for the sequence 5'-GACGTC-3'. The exact positions of the cuts within the Ssp52.?0/-recognition site were determined according to the enzymatic sequencing approach described in (3) An M13mp/8 derivative with an insert containing a Ssp5230I cleavage site was used for enzymatic sequencing reactions starting with a 5'-phosphorylated universal M13 sequencing primer. In a parallel reaction, the same primer [P]-endlabelled with T4 PNK and [7-P]ATP, was annealed to the template and the labelled primer was extended by treatment with Klenow enzyme and all four dNTPs through the Ssp5230I site. The double stranded DNA was used as substrate for Ssp5230I to produce an 5'-endlabelled DNA fragment comparable to the sequencing ladder. Samples were analyzed without or with (—/+) further incubation with T4 DNA polymerase and all four dNTPs by electrophoresis and subsequent autoradiography (Figure 2). In the Ssp5230I reaction the observed single band comigrated with T(5); after T4 DNAP treatment the observed band shift refers to G(l) of the recognition sequence 5'-GACGTC-3'.