DNA-BINDING DISCRIMINATION OF THE MURINE DNA CYTOSINE-C-5 METHYLTRANSFERASE

Mammalian DNA cytosine-C-5 methyltransferase modifies the CpG dinucleo tide in the context of many different genomic sequences. A rigorous DN A binding assay was developed for the murine enzyme and used to define how sequences flanking the CpG dinucleotide affect the stability of t he enzyme:DNA complex, Oligonucleotides containing a single CpG site f orm reversible 1:1 complexes with the enzyme that are sequence-specifi c. A guanine/cytosine-rich 30 base-pair sequence, a mimic of the GC-bo x cis-element, bound threefold more tightly than an adenine/thymine-ri ch sequence, a mimic of the cyclic AMP responsive element. However, th e binding discrimination between hemi-and unmethylated forms of these DNA substrates was small, as we previously observed at the K-m(DNA) le vel (Biochemistry, 35, 7308-7315 (1996)). Single-stranded substrates a re bound much more weakly than double-stranded DNA forms. An in vitro screening method was used to select for CpG flanking sequence preferen ces of the DNA methyltransferase from a large, divergent population of DNA substrates. After five iterative rounds of increasing selective p ressure, guanosine/cytosine-rich sequences were abundant and contribut ed to binding stabilization for at least 12 base-pairs on either side of a central CpG. Our results suggest a read-out of sequence-dependent conformational features, such as helical flexibility, minor groove di mensions and critical phosphate orientation and mobility, rather than interactions with specific bases over the course of two complete helic al turns. Thus, both studies reveal a preference for guanosine/cytosin e deoxynucleotides flanking the cognate CpG. The enzyme specificity fo r similar sequences in the genome may contribute to the in vivo functi ons of this vital enzyme. (C) 1998 Academic Press Limited.