Journal: Nucleic Acids Research

Article Title: Adjacent mutations in the archaeal Rad50 ABC ATPase D-loop disrupt allosteric regulation of ATP hydrolysis through different mechanisms

doi: 10.1093/nar/gkz1228

Figure Lengend Snippet: D-loop mutations affects the Mre11 nuclease activity. (A and B) Mre11 exonuclease activity for truncated MR NBD ( A ) and full-length MR ( B ) complexes with various Rad50 mutations. Exonuclease activity was determined with a 29-nt dsDNA containing a 2-AP probe at the second nucleotide from the 3′-end (Exo2) in the presence of 5 mM Mg 2+ (white bars); 1 mM Mn 2+ (gray bars); 1 mM Mn 2+ , 5 mM Mg 2+ and 1 mM ATP (black bars); or 1 mM Mn 2+ , 5 mM Mg 2+ and 1 mM AMP-PNP (red bars). Bars and error bars represent the average and standard deviation of three replicates. ( C ) Mre11 exonuclease activity for full-length MR complexes with various Rad50 mutations. Exonuclease activity was determined with a 29-nt dsDNA containing a 2-AP probe at the eleventh nucleotide from the 3′-end (Exo11) in the presence of 5 mM Mg 2+ (white bars); 1 mM Mn 2+ (gray bars); 1 mM Mn 2+ , 5 mM Mg 2+ , and 1 mM ATP (black bars); or 1 mM Mn 2+ , 5 mM Mg 2+ and 1 mM AMP-PNP (red bars). Bars and error bars represent the average and standard deviation of three replicates. (D and E) Mre11 endonuclease activity for full-length MR ( D ) and MR NBD ( E ) complexes with various Rad50 mutations. Endonuclease activity was measured using the ΦX174 single-stranded plasmid DNA in the absence (No Mn 2+ ) or presence of 1 mM Mn 2+ , 5 mM Mg 2+ and 1 mM ATP. (F and G) DNA end tethering assay for the various mutants in MR ( F ) and MR NBD ( G ) complexes. End tethering was assessed with linearized plasmid DNA in the presence of 2 mM ATP.

Article Snippet: Endonuclease reactions were performed using 1 μg of ΦX174 single stranded virion circular DNA (New England Biolabs) as the substrate in 30 μl reactions.

Techniques: Activity Assay, Standard Deviation, Plasmid Preparation