| DNA phosphorothioate modification exists in a wide variety of bacterial species. On a plasmid pIJ101 from Streptomyces lividans, a preferential sulfur-modification site was located within a 130-bp region with a 4-bp core sequence flanked by three direct repeats and two inverted repeats.An effort was made to target proteins that recognize the preferential modification region by fluorescent gel shift mobility assays. Three shift bands were observed. Their specificity of interaction with the site was confirmed by competition experiments. The three bands were also observed with protein sample from HXY6 (dnd-), indicating that the shift bands were not encoded by dnd genes.A series of protein purification procedures were performed to isolate proteins corresponding to the gel shift bands. After streptomycin precipitation, hydrophobic chromatography, anion exchange chromatography and native gradient gel electrophoresis, two homologue proteins from S.coelicolor were identified by mass spectrometry: they are polynucleotide phosphorylase (PNPase, SCO5737) and DNA Gyrase subunit A (GyraseA, SCO3873).To confirm the result of mass spectrometry, pnpase and gyraseA genes were cloned from S.lividans genome onto pET15b. Sequencing suggested that pnpase and gyraseA of S.lividans were identical to that of S.coelicolor. His-fusion proteins of PNPase and GyrA were expressed and purified to near homogeneity. Heterologously expressed PNPase showed similar shift band pattern and binding specificity to those of wild type PNPase, while recombinant GyraseA bound poorly to preferential modification sequence.Rabbit polyclonal antibody against PNPase was raised using recombinant protein. Supershift assay confirmed that PNPase was one of the three shift bands.PNPase was known to function in RNA metabolism, such as mRNA degradation, RNA 3'-tail polymerization, etc. Its biological function of specific binding to preferential DNA phosphorothioate modification sequence remains to be elucidated.pnpase was reported to be essential in S.coelicolor. We are using overexpression and antisense RNA gene silencing techniques to investigate its biological function. Further study on PNPase will provide insight into the function of DNA preferential phosphorothioation. |
