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In this paper we demonstrate that the HincII restriction endonuclease, in addition to being sensitive to methylation of the 3' A and C residues, is also sensitive to methylation of a cytosine immediately 5' to the recognition sequence. Having encountered this property in one of the sites in the mouse c-fos gene, we confirmed the sensitivity of HincII to the 5' cytosine methylation in in vitro methylated pUC12, pBR322 and p fos-1 plasmids. HincII is a six base-cutter which recognises the sequence GTPyPuAC, the cleavage site being between the Py/Pu. Its activity is known to be sensitive to the methylation of the A residue in the sequence ( 1). Recently, Bull et al. ( 2) used plasmid constructs having GTCGAC G sequence for HincII digestion and showed that the presence of methylated cytosine flanked by a G at the 3' end of recognition sequence inhibits HincII digestion. They also showed that methylation in the internal CpG did not affect the digestion. However, the HincII sensitivity to C methylation has not been universally accepted (see ref. 3). Here, we report that in the DNA motif C GTCGACC, HincII digestion is sensitive to methylation of the 5'C which is not a part of its recognition sequence. We also confirm that methylation of the internal C has no effect on the digestion by HincII.

Restriction enzyme HincII is sensitive to methylation of cytosine that occurs 5' to the recognition sequence