EcoP15I is a Type III
restriction endonuclease requiring the interaction with two inversely oriented 5'-CAGCAG
recognition sites for efficient
DNA cleavage. Diverse models have been developed to explain how
enzyme complexes bound to both sites move toward each other,
DNA translocation,
DNA looping and
simple diffusion along the
DNA. Conflicting data also exist about the impact of cofactor
S-adenosyl-L-methionine (
AdoMet), the
AdoMet analogue sinefungin and the bases flanking the
DNA recognition sequence on EcoP15I
enzyme activity. To clarify the functional role of these questionable parameters on EcoP15I activity and to optimize the
enzymatic reaction, we investigated the influence of cofactors, ionic conditions, bases flanking the
recognition sequence and
enzyme concentration. We found that
AdoMet is not necessary for
DNA cleavage. Moreover, the presence of
AdoMet dramatically
impaired DNA cleavage due to competing
DNA methylation. Sinefungin neither had an appreciable effect on
DNA cleavage by EcoP15I nor compensated for the second
recognition site. Moreover, we discovered that
adenine stretches on the
5' or
3' side of CAGCAG
led to preferred cleavage of this site. The length of the
adenine stretch was pivotal and had to be different on the two sides for most efficient cleavage. In the absence of
AdoMet and with
enzyme in
molar excess over
recognition sites, we observed minor cleavage at two communicating
DNA sites simultaneously. These results could also be exploited in the high-throughput, quantitative
transcriptome analysis method
SuperSAGE to optimize the crucial EcoP15I
digestion step.
