Interaction of the HpaI endonuclease with synthetic oligonucleotides.
To determine which functional groups of bases within the grooves of double-helical DNA interact with the HpaI endonuclease, we have employed chemically synthesized octanucleotides containing base analogues. The 5-methyl group of thymine was probed as a contact between the HpaI endonuclease and its recognition sequence by using the oligonucleotides d(G-G-T-T-A-A-C-C), d(G-G-T-U-A-A-C-C), and d(G-G-T-U(Br)-A-A-C-C). The 2-amino group of guanine was probed as a contact for the HpaI endonuclease by using the octanucleotide d(G-I-T-T-A-A-C-C). The HpaI endonuclease cleaves octanucleotides d(G-G-T-T-A-A-C-C) and d(G-G-T-B-A-A-C-C) according to Michaelis-Menten kinetics. However, both the Km and turnover number for d(G-G-T-B-A-A-C-C) were severalfold lower than those for cleavage of d(G-G-T-T-A-A-C-C). In addition, d(G-G-T-U-A-A-C-C) was not cleaved by HpaI endonuclease, suggesting that the 5-methyl group of thymine is a contact between the HpaI endonuclease and its recognition sequence. d(G-I-T-T-A-A-C-C) was not cleaved by the HpaI endonuclease which may be due in part to the low thermal stability of the duplex. Nevertheless, our results suggest that the 2-amino group of guanine is a contact for the HpaI endonuclease. A phosphate group 5' external to the HpaI recognition sequence has been identified as a contact between the HpaI endonuclease and DNA. The HpaI endonuclease cleaved 5'-phosphorylated octanucleotide 30-fold faster than unphosphorylated octanucleotide. In addition, the Km of the d(G-G-T-T-A-A-C-C) was 8000-fold higher than the Km of the phage f1 RFI DNA, suggesting that the octanucleotide is too short to take advantage of the entire DNA binding site of the enzyme.
D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004262:DNA Restriction Enzymes; C024406:endodeoxyribonuclease HpaI; D015252:Deoxyribonucleases, Type II Site-Specific