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. Author manuscript; available in PMC: 2015 May 25.
Published in final edited form as: Nat Commun. 2014 Nov 25;5:5506. doi: 10.1038/ncomms6506

Figure 4. Functional assays examining ATP hydrolysis and DNA processing by HerA-NurA.

Figure 4

(A) ATPase assays measuring free Pi release by the HerA-(D58A) NurA complex. Three DNA substrates were used: blunt ended dsDNA, or dsDNA with 5′ or 3′ single-stranded tails, respectively. Native HerA (WT), the Walker A mutant (K154A), the trans-acting arginine mutants (R142A and R381A), the putative DNA-binding loop mutant (K363A), the “brace” mutation (D471A), and the R142A/R381A, R142A/D471A and R381A/D471A mutants were examined. The data points represent the mean of three independent repeats and the error bars one standard deviation. (B) (Left) DNA processing assays of a 5.3 kb dsDNA substrate (using 2 μM HerA-NurA complex on 1.25 nM dsDNA at 60°C). Products were examined at five time-points: 0 (underlined), 5, 10, 20 and 40 minutes, following separation on a 0.8% agarose gel and visualisation by ethidium bromide staining. Lanes 1-5, WT HerA-WT NurA complex; lanes 6-10, K154A HerA-WT NurA; lanes 11-15, R142A HerA-WT NurA; lanes 16-20, R381A HerA-WT NurA; lanes 21-25, K363A HerA-WT NurA; lanes 26-30, D471A HerA-WT NurA. (Right) Assay quantification. The data points represent the mean of three independent repeats and the error bars one standard deviation. Additional controls are shown in Supplementary Fig. 4. (C) (Top) DNA unwinding activity by HerA-(D58A)NurA (0.46 μM HerA-NurA complex and 10 nM dsDNA, 5′ single-stranded overhanging, substrate) (16). After 30 mins at 60°C, products were resolved by 12% PAGE and visualised by autoradiography. The DNA was 32P radiolabelled on the 5′ end of the shorter strand (asterisk). Lane 1 (underlined), boiled (unwound) sample; lane 2, no protein control; Lanes 3-8, native (WT) HerA, and K154A, R142A, R381A, K363A and D471A mutant HerA, respectively (all in complex with D58A NurA). (Bottom) Assay quantification. The data points represent the mean of three independent repeats and the error bars one standard deviation (D) Triplex displacement assay. Lane 1 (underlined) boiled control (TFO; 32P radiolabelled triplex forming oligonucleotide [asterisk]); lane 2, no protein control; lanes 3-6, native (WT) HerA, and K154A, D471A and G465STOP mutant HerA, respectively (all in complex with D58A NurA). Products were resolved on a 1% agarose gel, and visualised by autoradiography.