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DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5′ half (CAT) and a canonical 3′ half (GTG). DraIII nicks the 3′ canonical half site at a faster rate than the 5′ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0038-z) contains supplementary material, which is available to authorized users.

Aiming at the problems of over stacking, warping deformation and rapid adjustment of layer thickness in electron beam additive manufacturing, the 3D printing slicing algorithm and partition scanning strategy for numerical control systems are studied. The GPU (graphics processing unit) is used to slice the 3D model, and the STL (stereolithography) file is calculated in parallel according to the normal vector and the vertex coordinates. The voxel information of the specified layer is dynamically obtained by adjusting the projection matrix to the slice height. The MS (marching squares) algorithm is used to extract the coordinate sequence of the binary image, and the ordered contour coordinates are output. In order to avoid shaking of the electron gun when the numerical control system is forming the microsegment straight line, and reduce metal overcrowding in the continuous curve C0, the NURBS (non-uniform rational b-splines) basis function is used to perform curve interpolation on the contour data. Aiming at the deformation problem of large block components in the forming process, a hexagonal partition and parallel line variable angle scanning technology is adopted, and an effective temperature and deformation control strategy is formed according to the European-distance planning scan order of each partition. The results show that the NURBS segmentation fits closer to the original polysurface cut line, and the error is reduced by 34.2% compared with the STL file slice data. As the number of triangular patches increases, the algorithm exhibits higher efficiency, STL files with 1,483,132 facets can be cut into 4488 layers in 89 s. The slicing algorithm involved in this research can be used as a general data processing algorithm for additive manufacturing technology to reduce the waiting time of the contour extraction process. Combined with the partition strategy, it can provide new ideas for the dynamic adjustment of layer thickness and deformation control in the forming process of large parts.