The 5′-GGATCC-3′ cleavage specificity of BamHl is increased to 5′-CCGGATCCGG-3′ by sequential double methylation with M · HpaII and M · BamHI

Abstract

Site-specific DNA methylation is known to block cleavage by a number of restriction endonucleases. We show that methylation at ‘non-canonical’ DNA modification sites can also block methylation by five of 13 DNA methyltransferases (MTases) tested. Furthermore, MTases and endonucleases that recognize the same nucleotide sequence can differ in their sensitivity to non-canonical methylation. In particular, BamHI endonuclease can cut 5′-GGATCm 5C efficiently, whereas M· BamHI cannot methylate this modified sequence. Methyltransferase/endonuclease pairs which differ in their sensitivity to non-canonical methylation can be exploited to generate rare DNA cleavage sites. For example, we show that M· HpaII, M· BamHI, and BamHI can be used sequentially in a three-step procedure to specifically cleave DNA at the 10-bp sequence 5′-CCGGATCCGG. Several highly selective DNA cutting strategies are made possible by these sequential double methylation-blocking reactions.