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The (cytosine-5) DNA methyltransferase M.Hhal causes its target cytosine base to be flipped completelyout of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.Hhal binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog, S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferasefor cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.

The cleavage specificity of R.Cfr9I was determined to be CCGGG whereas the methylation specificity of H.Cfr9I was C4mCCGGG. The action of HspI, HpaII, SmaI, XmaI and Cfr9l restriction endonudeases on an unmethylated parent d(GGACCCGGGTCC) dodecanucleotide duplex and a set of oligo-nuoleotide duplexes, containing all possible substitutions of either 4mC or 5mC for C in the CCCGGG sequence, was investigated. It was found that 4mC methylation. in contrast to 5mC, renders the CCCGGG site resistant to practically all the investigated endonuoleases. The cleavage of methylated substrates with restriction endonucleases is discussed.

The synthesis of N4-methyl-2'-deoxycytidine and its fully protected mononucleotlae, suitable for the oligonucleotide synthesis by phosphotriester method is described, A set of octanucleotides - d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG) and dodecanucleotides - d(GGACCCGGGTCC), dfGGA5mCCCGGGTCC). d(GGA4mCCCGGGTCC) has been synthesized in a solution. Physical characterization of the oligonucleotide duplexes by means of UV and CD spectrometry provides the evidence that 4mC similarly to 5mC favours the B↑Z transition, although both of these methylated cytosines inhibit the B↑A conformational change. N4-Methylcytosine in contrast to 5-methyIcytosine reduces the DMA double nellx thermal stability.

An enzymatic activity rendering DNA immune to the action of the Smal restriction endonuclease in the presence of S-adenosyl-L-methionine has been detected in Serratla marcescens Sb. This methylase, M.Smal, modifies the second cytosine residue of the substrate sequence CCCGGG yielding N4-methylcytosine.