Limits...
ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation.

Simons M, Diffin FM, Szczelkun MD - Nucleic Acids Res. (2014)

Bottom Line: RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome.Protein roadblocks did not activate HsdR proteolysis.We note that an EcoKI nuclease mutant still produces cell death in a clpx- strain, consistent with DNA damage induced by unregulated motor activity.

View Article: PubMed Central - PubMed

Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.

Show MeSH

Related in: MedlinePlus

Proteolysis by ClpXP is specific to HsdR and is dependent upon site-specific translocation by the EcoKI holoenzyme. (A) Western blot using the polyclonal HsdR antibody for 60-min EcoKI reactions on pLKS5 in the absence or presence of ClpXP. Extended exposure of the blot reveals cross reaction with the HsdM subunit. The graph shows quantitation of the blot. (B) Example western blots (break shows different gels) for 60-min reactions under a range of conditions, using, as indicated: supercoiled DNA (SC); DNA pre-methylated at the EcoKI site (Meth); a DNA without an EcoKI site (Zero); or no added DNA (None). Graphs show quantified data: standard deviation error bars from at least three repeat experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231737&req=5

Figure 3: Proteolysis by ClpXP is specific to HsdR and is dependent upon site-specific translocation by the EcoKI holoenzyme. (A) Western blot using the polyclonal HsdR antibody for 60-min EcoKI reactions on pLKS5 in the absence or presence of ClpXP. Extended exposure of the blot reveals cross reaction with the HsdM subunit. The graph shows quantitation of the blot. (B) Example western blots (break shows different gels) for 60-min reactions under a range of conditions, using, as indicated: supercoiled DNA (SC); DNA pre-methylated at the EcoKI site (Meth); a DNA without an EcoKI site (Zero); or no added DNA (None). Graphs show quantified data: standard deviation error bars from at least three repeat experiments.

Mentions: To show that the inhibition of EcoKI cleavage by ClpXP was due to proteolysis of the HsdR subunit, the same reaction samples were also analysed by western blotting using an EcoKI polyclonal antibody (kindly provided by Marie Weiserova, Institute of Microbiology, Prague) (58). In the presence of ClpXP, HsdR levels dropped to ∼30% after 1 h (Figure 2B). Intermediate species were not observed, consistent with complete proteolysis by ClpXP following a rate-limiting step. Degradation of the HsdM subunit was not observed (Figure 3A), consistent with the earlier observations in vivo that the ClpXP interaction is specific to the HsdR subunit. We note that the incomplete HsdR digestion may be because those HsdR that are irreversibly trapped on the cleaved DNA are in a different conformation that can no longer be recognized by ClpXP. Inhibition of DNA cleavage is likely to occur when the HsdR concentration falls below the threshold for forming the holoenzymes (9). However, the DNA cleavage rate in our in vitro assay is faster than the HsdR degradation rate. This suggests that the interaction of ClpXP with HsdR can inhibit the EcoKI cleavage reaction well before actual proteolysis occurs.


ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation.

Simons M, Diffin FM, Szczelkun MD - Nucleic Acids Res. (2014)

Proteolysis by ClpXP is specific to HsdR and is dependent upon site-specific translocation by the EcoKI holoenzyme. (A) Western blot using the polyclonal HsdR antibody for 60-min EcoKI reactions on pLKS5 in the absence or presence of ClpXP. Extended exposure of the blot reveals cross reaction with the HsdM subunit. The graph shows quantitation of the blot. (B) Example western blots (break shows different gels) for 60-min reactions under a range of conditions, using, as indicated: supercoiled DNA (SC); DNA pre-methylated at the EcoKI site (Meth); a DNA without an EcoKI site (Zero); or no added DNA (None). Graphs show quantified data: standard deviation error bars from at least three repeat experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231737&req=5

Figure 3: Proteolysis by ClpXP is specific to HsdR and is dependent upon site-specific translocation by the EcoKI holoenzyme. (A) Western blot using the polyclonal HsdR antibody for 60-min EcoKI reactions on pLKS5 in the absence or presence of ClpXP. Extended exposure of the blot reveals cross reaction with the HsdM subunit. The graph shows quantitation of the blot. (B) Example western blots (break shows different gels) for 60-min reactions under a range of conditions, using, as indicated: supercoiled DNA (SC); DNA pre-methylated at the EcoKI site (Meth); a DNA without an EcoKI site (Zero); or no added DNA (None). Graphs show quantified data: standard deviation error bars from at least three repeat experiments.
Mentions: To show that the inhibition of EcoKI cleavage by ClpXP was due to proteolysis of the HsdR subunit, the same reaction samples were also analysed by western blotting using an EcoKI polyclonal antibody (kindly provided by Marie Weiserova, Institute of Microbiology, Prague) (58). In the presence of ClpXP, HsdR levels dropped to ∼30% after 1 h (Figure 2B). Intermediate species were not observed, consistent with complete proteolysis by ClpXP following a rate-limiting step. Degradation of the HsdM subunit was not observed (Figure 3A), consistent with the earlier observations in vivo that the ClpXP interaction is specific to the HsdR subunit. We note that the incomplete HsdR digestion may be because those HsdR that are irreversibly trapped on the cleaved DNA are in a different conformation that can no longer be recognized by ClpXP. Inhibition of DNA cleavage is likely to occur when the HsdR concentration falls below the threshold for forming the holoenzymes (9). However, the DNA cleavage rate in our in vitro assay is faster than the HsdR degradation rate. This suggests that the interaction of ClpXP with HsdR can inhibit the EcoKI cleavage reaction well before actual proteolysis occurs.

Bottom Line: RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome.Protein roadblocks did not activate HsdR proteolysis.We note that an EcoKI nuclease mutant still produces cell death in a clpx- strain, consistent with DNA damage induced by unregulated motor activity.

View Article: PubMed Central - PubMed

Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus