Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase

Abstract

The pseudorabies virus (PRV) DNase gene has an open reading frame of 1476 nt, capable of coding a 492-residue protein. A previous study showed that PRV DNase is an alkaline exonuclease and endonuclease, exhibiting an Escherichia coli RecBCD-like catalytic function. To analyse its catalytic mechanism further, we constructed a set of clones truncated at the Nterminus or C-terminus of PRV DNase. The deleted mutants were expressed in E. coli with the use of pET expression vectors, then purified to homogeneity. Our results indicate that (1) the region spanning residues 274-492 exhibits a DNA-binding ability 7-fold that of the intact DNase ; (2) the N-terminal 62 residues and the C-terminal 39 residues have important roles in 3h-exonuclease activity, and (3) residues 63-453 are responsible for 5h-and 3h-exonuclease activities. Further chemical modification

Identification of three crucial histidine residues (His115, His132 and His297) in porcine deoxyribonuclease II

“…In terms of a functional mechanism, the strongly nucleophilic histidine residues in nucleases are often involved in catalytic hydrolysis of phosphodiester bonds [8]. Mutation of the conserved His 371 residue in members of the herpesviral DNase family (H371A) was found to abrogate nuclease activity, even though the mutants retained their DNA binding abilities [9]. Similarly, another apoptotic nuclease, caspase-activated DNase, was found to have several functionally relevant histidine residues [10].…”

Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

“…Histidine residue has been implicated in the active site of several nucleases, including colicin E9, Serratia nuclease, I- Ppo I, Vvn, and viral deoxyribonuclease (DNase) [ 13 , 25 , 29 , 30 ]. In order to determine whether the histidine residue was also responsible for the catalytic activity of human EndoG, we treated EndoG with various amounts of DEPC.…”

“…The chemical modification was performed as described previously [ 25 ]. Briefly, EndoG (0.2 pmol) was mixed with various amounts of diethyl pyrocarbonate (DEPC) (Sigma, St Louis, MO, USA) in 50 mM potassium phosphate buffer (pH 6.0) and incubated at 25°C for 30 min.…”

“…Site-directed mutagenesis was performed as described previously [ 25 ]. Uracil-containing ssDNA was prepared by transforming pET-EndoG into E. coli CJ236 strain, which lost its deoxyuridine triphosphate nucleotidohydrolase and uracil glycosylase activities.…”

Molecular and Biochemical Characterization of YeeF/YezG, a Polymorphic Toxin-Immunity Protein Pair From Bacillus subtilis

“…Cells co-expressing YeeF-CT and YezG had a similar growth curve profile as control cells (Figure 2A), indicating that coexpression of YezG protects against YeeF-CT mediated toxicity. Histidine is a catalytically important residue in several nucleases (Ho et al, 2000). Multiple sequence alignment of YeeF-CT with homologs in other bacteria suggest that His581 is highly conserved (Supplementary Figure S1).…”