The nuclease, Vvn, is a nonspecific periplasmic nuclease capable of digesting DNA and RNA. its sequence-independent acknowledgement of DNA and RNA. Based on the enzymeCsubstrate and enzymeCproduct structures observed in the mutant VvnCDNA crystals, Actinomycin D kinase activity assay a catalytic mechanism is definitely proposed. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and shows how non-specific DNA-binding proteins may identify DNA. colicins, such as ColE7 and ColE9 (Ku et al., 2002; Walker et al., 2002), which are secretory endonucleases Actinomycin D kinase activity assay that digest chromosomal DNA to destroy target cells, thereby enabling the host cells to have a better survival advantage during times of stress. Vvn from (Wu et al., 2001) belongs to a family of periplasmic or extracellular nucleases that represent another class of non-specific endonucleases which are also involved in protecting bacterial cells. This family of enzymes include Dns from (Focareta and Manning, 1987), NucM from (Moulard et al., 1993), EndoI from (Jekel and Wackernagel, 1995), and Dns and DnsH from (Chang et al., 1992; Dodd and Pemberton, 1996). These endonucleases all contain a signal peptide located at the N-terminus and eight strictly conserved cysteine residues. The signal peptides are cleaved off during the enzymes transportation from cytoplasm to periplasm, resulting in a mature protein of an average size of 25kDa (see Figure?Figure1).1). All of these endonucleases are capable of digesting both DNA and RNA and are only active in their oxidized form (Wu et al., 2001). Some of them are extracellular endonucleases, such as the Dns from mutants lacking (encoding EndoI) or mutants lacking can be transformed more efficiently, but they resemble wild-type cells with regards to growth rate, conjugational properties and their ability to propagate various phages (Durwald and Hoffmann-Berling, 1968; Wu et al., 2001). The and mutants showed no observed virulence effects in the mouse model (Focareta and Manning, 1991; Wu et al., 2001), indicating that Dns and Vvn were not required for the bacterial virulence. Open in a separate window Fig. 1. Sequence alignment of Vvn (Wu et al., 2001) with several representative extracellular nucleases: Dns from (Focareta and Manning, 1987), Actinomycin D kinase activity assay NucM from (Moulard et al., 1993), EndoI from (Jekel and Wackernagel, 1995), and Dns and DnsH from (Chang et al., 1992; Dodd and Pemberton, 1996). The N-terminal signal peptide from residues 1 to 18 marked in the yellow-shaded box is cleaved off during transportation. The secondary structural elements of -helices and -strands in the crystal structure of Vvn are illustrated above the sequences. The eight cysteine residues making the four disulfide bridges are marked in blue. The residues responsible for metal binding are marked in red. Here we report the crystal structures of Vvn and a Vvn mutant in complex with an 8bp DNA, both at a resolution of 2.3?. Vvn represents the first solved structure in this family of extracellular endonucleases and it bears a novel mixed / topology. Comparison with other nucleases interestingly reveals a similar structural arrangement in the endonuclease active site containing two -strands and one -helix with a centrally located divalent metal ion. In the crystal structure of the VvnCDNA complex, an enzymeCsubstrate complex with an intact DNA and an enzymeCproduct complex with a cleaved DNA are both present, providing an ideal case to elucidate the hydrolysis mechanism. Moreover, the interaction between Vvn and DNA demonstrates how a protein may bind duplex DNA without sequence preference. Results Endonuclease activity The gene of was subcloned into pTYB2 vector and Vvn was overexpressed in B834. The purified Vvn had a mol. wt of 24780Da as measured by mass spectrometry, close to the calculated Mr of 24785Da for the matured Vvn containing residues 19C231 without the N-terminal signal peptide. A plasmid DNA was used as the substrate to monitor Vvn endonuclease activity through DNA topological changes in agarose gel electrophoresis. We found that the supercoiled DNA was cleaved into linear and open-circular forms or completely degraded with higher concentrations of enzymes. In identical buffer solutions (50mM TrisCHCl and 10mM MgCl2 at pH8), Vvn appeared to have a slightly better endonuclease activity than DNaseI and ColE7, but weaker activity than nuclease (see Figure?Figure22A). Open in a separate window Fig. 2. Characterization of the endonuclease activity of Vvn. (A)Comparison of the endonuclease activity of Vvn with different non-specific endonucleases. Several endonucleases in the concentration range of 0.01nMC0.1M were each incubated with a Slc3a2 plasmid DNA in the same buffer solution containing 10mM MgCl2 and 50mM TrisCHCl at pH8.0. The topological changes of the substrate DNA were monitored by 1% agarose gel electrophoresis. The supercoiled (s) form of the plasmid DNA was digested into open-circular (o) and linear (l) forms. In the response buffers used right here, Vvn made an appearance as a weaker DNase compared to the nuclease, but more vigorous than.