A DODECAPEPTIDE COMPRISING THE EXTENDED CHAIN-ALPHA-4 REGION OF THE RESTRICTION-ENDONUCLEASE ECORI SPECIFICALLY BINDS TO THE ECORI RECOGNITION SITE

The restriction endonuclease EcoRI binds and cleaves DNA containing GA ATTC sequences with high specificity. According to the crystal structu re, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha 4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDG MAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, Ec oRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at s ites that partially overlap with EcoRI sites. The peptide inhibits cle avage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. I t interferes with DNA methylation by the EcoRI methyltransferase but n ot by the BamHI methyltransferase. It competes with EcoRI for DNA bind ing. Based on these results, the DNA binding constant of the peptide t o GAATTC sequences was calculated to be 3 x 10(4) M(-1). DNA binding i s not temperature-dependent, suggesting that binding of the peptide is entropy driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in sp ite of the fact that the affinity is much smaller. These results sugge st that contacts to the phosphate groups in EcoRI mainly provide bindi ng affinity, whereas the specificity of EcoRI is based to a large ex t ent on sequence-specific base contacts.