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

Abstract

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.