Jeltsch A, Alves J, Urbanke C, Maass G, Eckstein H, Lianshan Z, Bayer E, Pingoud A
Institut für Biochemie, Justus-Liebig Universität, Giessen, Germany.
J Biol Chem. 1995 Mar 10;270(10):5122-9. doi: 10.1074/jbc.270.10.5122.
The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, 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 (WDGMAAGNAIER), 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, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 x 10(4) M-1. DNA binding is 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 spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.
限制性内切酶EcoRI能高度特异性地结合并切割含有GAATTC序列的DNA。根据晶体结构，该酶与DNA的大多数特异性接触是由延伸链区域和α-螺旋α4的第一圈（氨基酸137 - 145）形成的。在此，我们证明一种十二肽（WDGMAAGNAIER），其序列中带下划线部分与EcoRI的氨基酸137 - 145相同，能特异性结合GAATTC序列。该肽抑制EcoRI对DNA的切割，但不抑制BamHI、BclI、EcoRV、HindIII、PacI和XbaI的切割。XbaI在与EcoRI位点部分重叠的位点处切割DNA的速度会减慢。该肽抑制ApoI对GAATTC位点的切割，ApoI识别序列RAATTY。它干扰EcoRI甲基转移酶介导的DNA甲基化，但不干扰BamHI甲基转移酶介导的甲基化。它与EcoRI竞争DNA结合。基于这些结果，计算出该肽与GAATTC序列的DNA结合常数为3×10⁴ M⁻¹。DNA结合不依赖温度，这表明该肽的结合是由熵驱动的。由于该肽对DNA没有任何非特异性结合，尽管其亲和力小得多，但其DNA结合特异性与EcoRI相似。这些结果表明，EcoRI中与磷酸基团的接触主要提供结合亲和力，而EcoRI的特异性在很大程度上基于序列特异性碱基接触。
