Deoxyribonuclease crystallization

The reasons for selecting pancreatic DNase I as one of the two representative of mammalian DNases are to a large extent historical. Deoxyribonuclease I was the first enzyme to be recognized as specific for DNA (18-15), the first DNase to produce 5 -monoesterified products (16, 17), the first DNase to be crystallized (18), the first DNase to have a specific protein inhibitor (19-23), the first DNase shown to produce nicks on one strand in preference to scission of both strands (24, 25). A new first has been added recently (25a) DNase I was covalently coupled to porous glass, thus supplying an insoluble DNase. 

L17. Lindberg, M. U., Crystallization from calf spleen of two inhibitors of deoxyribonuclease. J. Biol. Chem. 241, 1246-1249 (1966). 

At the level of primary structure, several recent experiments have shown the effect of base sequence on the local structure of DNA. A dramatic example is the crystal structure of d(CpG) as determined by Rich and coworkers ( ). This molecule crystallizes in a left-handed double helical form called Z-DNA, which is radically different in its structural properties from the familiar right-handed B-DNA structure. Dickerson and Drew (10) showed in the crystal structure of the dodecanucleotide d(CGCGAATTCGCG) that the local twist angle of a DNA double helix varies with sequence. Deoxyribonuclease I cuts the phosphodiester backbone of the dodecanucleotide preferentially at sites of high twist angle (l 1). From these and other (12,13) experiments we see that the structure of DNA varies with base sequence, and that enzymes are sensitive to these details of structure. 

Because of its tendency to polymerize, G-actin has been difficult to crystallize. However, it forms crystalline complexes with several other proteins, e.g., deoxyribonuclease 1, a fragment of gelsolin, and profilin, which block polymerizafion and if has recently been crystallized as the free ADP complex. The fhree-dimensional structure of the actin is nearly the same in all cases. The molecule folds into four domains, the ATP binding site being buried in a deep cleft. The atomic structure (Fig. 7-10) resembles that of hexo-kinase, of glycerol kinase, and of an ATP-binding domain of a chaperonin of fhe Hsp 70 family. As with the kinases, actin can exist in a closed and more open conformations, one of which is seen in the profilin complex. Addition of 1 mM Mg + or 0.1 M KCl to a solution of G-actin leads to spontaneous transformation into filaments of F-actin (Figs. 7-10 and... 

The role of the divalent metal ions present in natural phosphodiesterases became clear in bovine pancreatic deoxyribonuclease I (DNase I), the first endonuclease structure determined by X-ray crystallography. The nucleophilic attack of a water molecule activated by a histidine residue is facilitated by the interaction of a calcium ion with the phosphate group to be cleaved (291). Glutamic and aspartic residues involved in magnesium binding have been identified in the crystal structure of four type II restriction enzymes EcoRl (292), EcoRV (293), Pvull (294), and BamHl (295), as well as in that of the repair... 

Among the contaminants of DNA preparations obtained from mammalian tissue, protein and RNA are the most conspicuous. Proteins can be separated by denaturation with chloroform and amyl alcohol, a procedure that may be followed by repeated washings with sodium dodecyl sulfate. RNA can be eliminated by repeated digestion of the preparation with ribonu-clease. (Even crystallized RNase has deoxyribonuclease activity therefore, the RNase preparation must be boiled for 3 minutes before using it for treating the DNA preparation.)... 

Deoxyribonucleases. The enzyme from the pancreas has been studied best and has been crystallized its molecular weight is 60,000 the pH optimum is around 6.0 to 7.0. It splits the 3 -phosphoester bond, freeing oligonucleotides. There remain also some nondialyzable, large fragments. 

---