journal menu
![[Figure 6]](mn5067fig6mag.jpg)
|
|
Figure 6
Imidazole chemical rescue assays for nuclease activity in GBS_NucA mutants. (a) Nuclease-activity assays for GBS_NucA mutants on the background of H148G. The DNA substrate used for this assay is a supercoiled plasmid, which is then nicked to the open-circle form. The open-circle form accumulates double-strand breaks, linearizing the plasmid, and is further degraded to lower molecular-weight fragments. (b) The percentage of each form was calculated using ImageQuant TL (GE Healthcare) and the standard deviation for each calculation is indicated by the error bars. All reactions were performed in triplicate. (c) Single radial enzyme diffusion (SRED) assay for GBS_NucA mutants performed in the presence of 30 mM imidazole. The extent of nuclease degradation is indicated by the size of the `halo' and results from a fluorescence decrease correlated to a decrease in double-stranded DNA substrate. (d) Nuclease activity for each GBS_NucA mutant was calculated by measuring the `halo' radius, normalizing to the amount of activity of the H148G mutant (calculated as 100%). All reactions were performed in triplicate, with the error bars indicating the standard deviation for each calculation. |
![[Figure 6]](mn5067fig6.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
Open 
access
access
