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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.

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Proteolysis of HsdR is dependent on translocation and not DNA cleavage. (A) Quantified data from 60-min reactions on pLKS5 in the presence or absence of ClpXP, and with EcoKI MTase without HsdR, with wild-type HsdR or with HsdRs mutated in either a nuclease (Nuc-) or ATPase (ATP-) motif. Standard deviation error bars from three repeat experiments. (B) Transformation screen to measure RA by ClpXP. Colony forming units (c.f.u.) were measured in separate transformation reactions using pBR322 or plasmids carrying the complete EcoKI operon (WT), the genes for the MTase alone (ΔHsdR) or the EcoKI operon with an HsdR mutated within the nuclease domain (D298E). The first two rows show the quotient of the c.f.u. for the EcoKI plasmid and pBR322 in strains with active ClpXP (NK311) or without active ClpXP (NK312). The third row is the quotient of the transformation efficiency in the presence and absence of ClpXP, with elevated values indicating ClpXP-dependent RA. The grey box indicates that plasmid preparations from the successful transformants showed loss of DNA in 100% of cases examined. The smaller DNA could retransform NK312 more efficiently than the original DNA, indicating loss of EcoKI activity.
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Figure 4: Proteolysis of HsdR is dependent on translocation and not DNA cleavage. (A) Quantified data from 60-min reactions on pLKS5 in the presence or absence of ClpXP, and with EcoKI MTase without HsdR, with wild-type HsdR or with HsdRs mutated in either a nuclease (Nuc-) or ATPase (ATP-) motif. Standard deviation error bars from three repeat experiments. (B) Transformation screen to measure RA by ClpXP. Colony forming units (c.f.u.) were measured in separate transformation reactions using pBR322 or plasmids carrying the complete EcoKI operon (WT), the genes for the MTase alone (ΔHsdR) or the EcoKI operon with an HsdR mutated within the nuclease domain (D298E). The first two rows show the quotient of the c.f.u. for the EcoKI plasmid and pBR322 in strains with active ClpXP (NK311) or without active ClpXP (NK312). The third row is the quotient of the transformation efficiency in the presence and absence of ClpXP, with elevated values indicating ClpXP-dependent RA. The grey box indicates that plasmid preparations from the successful transformants showed loss of DNA in 100% of cases examined. The smaller DNA could retransform NK312 more efficiently than the original DNA, indicating loss of EcoKI activity.

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).


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 of HsdR is dependent on translocation and not DNA cleavage. (A) Quantified data from 60-min reactions on pLKS5 in the presence or absence of ClpXP, and with EcoKI MTase without HsdR, with wild-type HsdR or with HsdRs mutated in either a nuclease (Nuc-) or ATPase (ATP-) motif. Standard deviation error bars from three repeat experiments. (B) Transformation screen to measure RA by ClpXP. Colony forming units (c.f.u.) were measured in separate transformation reactions using pBR322 or plasmids carrying the complete EcoKI operon (WT), the genes for the MTase alone (ΔHsdR) or the EcoKI operon with an HsdR mutated within the nuclease domain (D298E). The first two rows show the quotient of the c.f.u. for the EcoKI plasmid and pBR322 in strains with active ClpXP (NK311) or without active ClpXP (NK312). The third row is the quotient of the transformation efficiency in the presence and absence of ClpXP, with elevated values indicating ClpXP-dependent RA. The grey box indicates that plasmid preparations from the successful transformants showed loss of DNA in 100% of cases examined. The smaller DNA could retransform NK312 more efficiently than the original DNA, indicating loss of EcoKI activity.
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Related In: Results  -  Collection

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Figure 4: Proteolysis of HsdR is dependent on translocation and not DNA cleavage. (A) Quantified data from 60-min reactions on pLKS5 in the presence or absence of ClpXP, and with EcoKI MTase without HsdR, with wild-type HsdR or with HsdRs mutated in either a nuclease (Nuc-) or ATPase (ATP-) motif. Standard deviation error bars from three repeat experiments. (B) Transformation screen to measure RA by ClpXP. Colony forming units (c.f.u.) were measured in separate transformation reactions using pBR322 or plasmids carrying the complete EcoKI operon (WT), the genes for the MTase alone (ΔHsdR) or the EcoKI operon with an HsdR mutated within the nuclease domain (D298E). The first two rows show the quotient of the c.f.u. for the EcoKI plasmid and pBR322 in strains with active ClpXP (NK311) or without active ClpXP (NK312). The third row is the quotient of the transformation efficiency in the presence and absence of ClpXP, with elevated values indicating ClpXP-dependent RA. The grey box indicates that plasmid preparations from the successful transformants showed loss of DNA in 100% of cases examined. The smaller DNA could retransform NK312 more efficiently than the original DNA, indicating loss of EcoKI activity.
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).

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