CRYSTAl structures OF restriction endonuclease eCOO109i

Hiroshi Hashimoto,^a Tsuyoshi Imasaki,^a Matsuri Kato,^a Toshiyuki Shimuzu,^a Mamoru Sato,^a and Keiko Kita^b

^aGraduate School of Integrated Science, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan; ^bGraduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan (hash@tsurumi.yokohama-cu.ac.jp)

Restriction endonucleases in bacteria recognize and cleave foreign DNA that is unmethylated. Most of the > 3,000 restriction endonucleases discovered to date belong to the type II class. The orthodox type II restriction endonuclease is homodimer, which recognize short and usually palindoromic sequences of 4~8 base pairs, and in the presence of Mg²⁺ cleaves the DNA within or immediately adjacent to the recognition site to give a 5’-phospahte and a 3’-OH end. Although type II restriction endonucleases are widely spread within bacteria, there is generally no sequence similarity among them. Type II restriction endonucleases comprise the largest family of functionally related enzymes, but less known about how their specificity.

The restriction endonuclease, EcoO109I from Escherichia coli forms functional homodimer and its molecular weight is 62 kDa. The EcoO109I monomer is made up with 272 amino acids. EcoO109I recognizes and cleaves the interrupted DNA sequence of 5’-RG|GNCCY-3’ and produces 5’-overhang DNA. To understand the molecular basis of DNA recognition and hydrolysis of phosphodiester bond, we have studied crystallographic analysis of EcoO109I.

Crystal of EcoO109I (DNA free) was obtained by hanging drop vapour diffusion method using polyethylene glycol as a precipitant at 277 K. Crystal of EcoO109I (DNA free) belongs to space group of I4 with the unit cell parameters of a = b = 175.5 Å, c = 44.6 Å. X-ray diffraction experiments for native data collection of EcoO109I DNA free crystals were performed with ACSC Quantum 4R CCD deterctor at BL38B1, SPring-8. The intensity data consists of 307,433 measurements of 26,765 unique reflections with an overall R_merge = 4.2% and 99.1% of theoretically observable reflections at 2.4 Å resolution. Crystal structure of EcoO109I was determined by MAD method with mercury derivative crystal using programs SOLVE/RESOLVE. The three-wavelength MAD data were collected by Rigaku MSC Jupiter 210 CCD detector at BL45PX, SPring-8.

Crystal of EcoO109I DNA complex was obtained by hanging drop vapour diffusion method using polyethylene glycol as a precipitant at 293 K. Crystal of EcoO109I DNA complex belongs to space group of P2₁2₁2₁ with the unit cell parameters of a = 71.8 Å, b = 203.2 Å, and c = 49.1 Å. Diffraction intensities of native and mercury derivative crystals of DNA complex were collected by Rigaku R-AXIS IV⁺⁺ imaging plate detector on Rigaku FR-D rotating anode X-ray generator. The native diffraction data consists of 202,201 measurements of 55,952 unique reflections with an overall R_merge = 8.8% and 93.5% of theoretically observable reflections at 1.9 Å resolution. Crystal structure of the DNA complex was determined by SIRAS method using programs SOLVE/RESOLVE.

Crystallographic structure refinement of EcoO109I and DNA complex are now in progress.