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.