Functional Characterization and Modulation of the DNA Cleavage Efficiency of Type III Restriction Endonuclease EcoP15I in Its Interaction with Two Sites in the DNA Target

Optimized design and data analysis of tag-based cytosine methylation assays

“…In order to evaluate the yield of desired products, we counted the number of reads containing this sequence and plotted the starting positions of this sequence within the reads obtained. We observed that approximately two-thirds of the reads contained the expected sequence, and found that the majority was located at base positions 25 and 26, consistent with the known digestion properties of the restriction enzyme [19]. Removal of the approximately 30% of reads lacking the CG-EcoP15I sequence was performed to eliminate spurious sequences.…”

“…Our protocol does not require gel extraction and only begins to show an under-representation of sequences when the (G+C) content exceeds approximately 80% (Figure 4a). We also tested to see whether the sizes of the MspI fragments generated influenced the counts obtained, as the digestion by type III endonucleases like EcoP15I is most efficient when a pair of enzymes is present in convergent orientation on the same DNA molecule [19]. We find that there is indeed an over-representation for shorter (≤300 bp) and a corresponding modest under-representation for larger MspI fragments (Figure 4b).…”

“…Apart from a modest decrease in representation in regions above approximately 80% (G+C) content, base composition did not cause biases in representations, possibly in part due to our avoidance of a gel purification step in library preparation [22]. Fragment length does influence the outcome, most likely due to effects on EcoP15I digestion [19], although the effects should be similar for both HpaII and MspI and should, therefore, largely cancel each other out in the normalization step. It is possible that endogeneous EcoP15I sites could influence the representations, but to have an effect they would have to be located within the 27 bp adjacent to HpaII/MspI sites and would cause digestion of the ligated adapter, causing those loci to be under-represented in both HpaII and MspI datasets.…”

DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements

“…The TLC model has difficulty in accounting for this cleavage. Effective cleavage of circular single-site substrates (25), of linear single-site substrates (26–28), of linear substrates with two sites in a tail-to-tail orientation (14,28) and of substrates where the two sites actually contact each other (28) have all been observed. Furthermore, cleavage of linear versions of the head-to-head substrates was enhanced to the levels seen with circular substrates if the free ends of the DNA were blocked by a streptavidin molecule (14,29).…”

“…The MT model has been put forward most forcefully by Szczelkun et al (30), but the model disregards the loops observed by AFM and other biochemical evidence for looping, such as the obvious cleavage of single-site circular DNA (25,29) and the observation that cleavage is hindered by supercoiling of the DNA (19). The absence of apparent looping in the TPM assay when applied to the type III R/M enzymes is also cited as support for the MT model (13,14), but it is apparent that the data are not of sufficient resolution, when compared with the data obtained for the Ecl18kI type II restriction enzyme, to rule out looping (32).…”