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.
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.
Möncke-Buchner
Elisabeth
E
Institute of Medical Virology, Helmut-Ruska-Haus, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Rothenberg
Maja
M
Reich
Stefanie
S
Wagenführ
Katja
K
Matsumura
Hideo
H
Terauchi
Ryohei
R
Krüger
Detlev H
DH
Reuter
Monika
M
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015263:Deoxyribonucleases, Type III Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020869:Gene Expression Profiling; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D012436:S-Adenosylmethionine; C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific; D000241:Adenosine; C006235:sinefungin