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Figure 2 | BMC Molecular Biology

Figure 2

From: Efficient 5′-3′ DNA end resection by HerA and NurA is essential for cell viability in the crenarchaeon Sulfolobus islandicus

Figure 2

A HerA(D176E) mutant with reduced ATPase activity is able to complement deletion of the wild-type herA gene. (A) A schematic map depicting the procedures of the complementation analysis. pMID-herA-T cells were transformed with pSSR vectors. The cells were selected in the presence of simvastatin. Counter-selection was performed with solid medium containing 5-FOA, uracil, and simvastatin. Two genotypes of the cell grown up were indicated as and . The star indicates a putative spontaneous mutation in pyrEF. (B) Genotype analysis of cells by PCR using herA flanking primers. The genomic DNA of each strain was isolated and used as the template. The pSSR plasmids in cells were isolated and amplified in E. coli DH5α for sequencing. Left panel, a schematic map showing the lengths of PCR products of different strains. Right panel, agarose gel electrophoresis of the PCR products. “+”, the designed plasmid maintained. (C) ATPase activity of HerA alone or with NurA in the absence or presence of DNA. The assay was performed as described in the Methods. Reaction products were separated using thin layer chromatography and analyzed by Phosphorimager. The means ± standard errors of three independent experiments and p values (n = 3) are shown. (D) ATPase activity of wild-type and mutant HerA proteins. The means ± standard deviations of three independent experiments are shown.

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