Figure 4

Purification of a putative PNKP from D. radiodurans and analysis of its 5' kinase and 3' phosphatase activities. A. Scheme of PNKP from D. radiodurans and H. sapiens. Protein domains are depicted according to predictions based on sequence similarities [36]. Schemes are not drawn to scale. HD, HD domain, kinase, polynucleotide kinase domain, HisB, histidinol phosphatase and related phosphatases domain. B. 3 μg of either D. radiodurans PNKP wt or PNKP R371K mutant were loaded onto a 10% SDS-PAGE and subsequently stained with Coomassie Blue R250. Both proteins were purified over 3 columns. Details are described in Methods. C. Titration of the D. radiodurans PNKP wt and PNKP R371K mutant to compare their polynucleotide kinase activity on a 5'OH 25 mer deoxyribose oligonucleotide. Different amounts of enzyme were incubated with the DNA substrate and γ-[³²P]-ATP as described in Methods. [³²P]-labelled 25 mer was detected by autoradiography.