Expression, purification and crystallization of two endonuclease III enzymes fromDeinococcus radiodurans

Abstract

Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiationresistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Á76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å , = 89.34 and one molecule per asymmetric unit. The EndoIII-3Á76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å , = 102.53 and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Á76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

Structural and functional characterization of two unusual endonuclease III enzymes from Deinococcus radiodurans

“…The pure brown/orange coloured proteins were concentrated and stored at À80°C. The presence of the [4Fe-4S] cluster was confirmed by UV-vis spectroscopy (Sarre et al, 2014) and the purity, as judged by SDS-PAGE analysis, was estimated to be greater than 95%.…”

“…As described previously (Sarre et al, 2014), brownish crystals of DrEndoIII1 and DrEndoIII3D76 were obtained and X-ray diffraction data were collected on the ID23-1 beamline at the European Synchrotron Radiation Facility (ESRF; Grenoble, France) (Nurizzo et al, 2006). The structure of DrEndoIII1 was solved by molecular replacement using MOLREP (Vagin and Teplyakov, 2010) and PHASER (McCoy et al, 2007) and the E. coli EndoIII as a search model (PDB ID: 2abk), while DrEndoIII3D76 was solved by the single-wavelength anomalous dispersion (SAD) method making use of the intrinsic anomalous signal from the [4Fe-4S] cluster (Sarre et al, 2014) (Table 1).…”

“…For the structural studies, DrEndoIII1, with a non-cleavable his-tag and an N-terminally truncated DrEndoIII3 (DrEndoIII3D76) were expressed, purified and crystallized as described previously (Sarre et al, 2014). For the functional studies DrEndoIII1, DrEndoIII2, DrEndoIII3 and DrEndoIII3D76 were expressed in BL21(DE3)pLysS at 20°C overnight with cleavable N-terminal his-tags allowing removal of the his-tags to make sure they do not interfere with the activity measurements.…”

“…The three genes encoding endonuclease III enzymes, DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0928 (DrEndoIII3), were amplified by PCR from D. radiodurans genomic DNA and inserted into pDest14 (Invitrogen) as described previously (Sarre et al, 2014). For the structural studies, DrEndoIII1, with a non-cleavable his-tag and an N-terminally truncated DrEndoIII3 (DrEndoIII3D76) were expressed, purified and crystallized as described previously (Sarre et al, 2014).…”

“…coli EndoIII (EcEndoIII) was cloned into the pDest14 expression vector and expressed as for the DrEndoIII enzymes (Sarre et al, 2014). EcEndoIII was purified according to the protocol for DrEndoIII2 but with 500 mM NaCl and 5 mM b-mercaptoethanol in the buffers, and all the purification steps were carried out at 4°C to minimise precipitation.…”