ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation.
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.
Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.Show MeSH
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Mentions: To explore the consistency between our in vitro results and the previous in vivo observations, we also examined the effect of two different HsdR mutants on ClpXP activity (Figure 4). HsdR(D298E) is mutated in Motif II of the RecB-family nuclease domain. A holoenzyme formed with this HsdR (Nuc-) cannot cleave DNA but can hydrolyze ATP and translocate similarly to wild-type HsdR (15,59,60). HsdR(K477R) is mutated in Motif I/Walker A box of the Superfamily 2 helicase domain. A holoenzyme formed with this HsdR (ATP-) cannot hydrolyze ATP, and thus neither translocates on nor cleaves DNA (61,62). Neither mutant could cleave DNA above background (Figure 4A, upper panel). HsdR(D298E) was still degraded by ClpXP, whilst HsdR(K477R) was not degraded, within error (Figure 4A, lower panel). These results match the observations in vivo with the same mutants (24,26) and are consistent with a requirement for DNA translocation rather than DNA cleavage (42).
Affiliation: DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.