Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis

Abstract

The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity. Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA protein), and 28 kDa. Only the AddB and AddA proteins are required for ATP-dependent exonuclease activity. Both the AddB and AddA proteins contained a conserved amino acid sequence for ATP binding. In the AddA protein, a number of small regions were present showing a high degree of sequence similarity with regions in the E. coli RecB protein. The AddA protein contained six conserved motifs which were also present in the E. coli helicase II (UvrD protein) and the Rep helicase, suggesting that these motifs are involved in the DNA unwinding activity of the enzyme. When linked to the T7 promoter, a high level of expression was obtained in E. coli.