Rapid and reliable method for identification of associated endonuclease cleavage and recognition sites.
One barrier to cross during genetic engineering is the restriction-modification system found in many bacteria. In this study, we developed a fast and reliable method for mapping the recognition and cleavage site of the restriction endonucleases. Clostridium pasteurianum, a model organism for the study of nitrogen fixation, has been found to harbour at least two restriction-modification systems including the restriction endonucleases CpaPI, which is an isoschizomer of MboI and CpaAI. Dam-methylated DNA was used to isolate the activity of CpaAI. Exposing freshly prepared cell lysate to known nucleotide fragments and directly sequencing the pool of digested nucleotide fragments enabled identification of the cleavage sites in the fragments. By aligning the sequences adjacent to the cleavage site, it was possible to identify the recognition sequence. Using this method, we successfully located all CpaAI recognition and cleavage sites within the template sequence. By modifying DNA with both Dam and CpG methylases (M.SssI) and thereby preventing digestion by CpaPI and CpaAI, no further endonuclease activity was detected.


CONCLUSIONS
Restriction-modification systems are important barriers to successful genetic modification in many bacterial species. In this study, we demonstrate an efficient and general applicable method for identifying endonuclease recognition and cleavage sites. For the study and the trails, the model organism for nitrogen fixation Clostridium pasteurianum was used. The method was proven to be reliable, and by modifying DNA at the identified sites, it is possible to prevent digestion.
Section for Sustainable Biotechnology, Aalborg University Copenhagen, Copenhagen, Denmark.
T Ø
TØ
Jensen
T
T
Kvist
M J
MJ
Mikkelsen
P
P
Westermann
D001483:Base Sequence; D001665:Binding Sites; D003013:Clostridium / Q000235:genetics; D053837:DNA Cleavage; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids; D015203:Reproducibility of Results; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D009841:Oligonucleotides; D004262:DNA Restriction Enzymes
Clostridium; methylation; sequencing