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Abstract
EcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod2 dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597–610.). Surprisingly the Mod2 enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res1Mod2 enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod2 core by the Res subunit.
Highlights
Since its introduction by Ward [1], 2-aminopurine (2AP) has proven to be a versatile probe of DNA and RNA structure and of the interaction of proteins with these polymers. This is because its fluorescence emission is intense, whereas the canonical bases are practically non-fluorescent, its excitation wavelength is longer than that of the canonical bases, enabling its selective excitation, and, most importantly, it has great sensitivity to its immediate environment [2,3,4,5,6,7]. 2AP fluorescence is normally highly quenched when it is stacked in a DNA double helix, when electron transfer quenching mechanisms involving nearby guanine bases are active, but any structural perturbation can dramatically enhance its emission. This is informative when using time-correlated single photon counting (TCSPC) to examine the fluorescence decay of 2AP, as demonstrated in previous studies of the nucleotide flipping mechanism used by DNA methyltransferases (MTases) [8,9,10,11,12], restriction endonucleases [13], DNA repair
The subunit stoichiometry of EcoP15I has been reported as being either Res1Mod2 or Res2Mod2 [27,28,29,30]. This discrepancy will make the determination of protein concentration and the extent of DNA binding difficult so we first investigated the subunit composition of our sample of EcoP15I
The clear conclusion from our work is that only the base at the methylation site, the second adenine in the target sequence of EcoP15I, is flipped by the Res1Mod2 RM enzyme
Summary
Since its introduction by Ward [1], 2-aminopurine (2AP) has proven to be a versatile probe of DNA and RNA structure and of the interaction of proteins with these polymers This is because its fluorescence emission is intense, whereas the canonical bases are practically non-fluorescent, its excitation wavelength is longer than that of the canonical bases, enabling its selective excitation, and, most importantly, it has great sensitivity to its immediate environment [2,3,4,5,6,7]. Flipping of the base induced by the enzyme greatly enhances the fractional amplitude of the long lifetime component at the expense of the short lifetime component and this change in amplitudes is a marker for base flipping
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