Limits...
The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I.

Sisáková E, Weiserová M, Dekker C, Seidel R, Szczelkun MD - J. Mol. Biol. (2008)

Bottom Line: We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain.In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics.The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

Show MeSH

Related in: MedlinePlus

DNA cleavage activity of motif II and III nuclease mutants of EcoR124I. (a) Comparison of cleavage end points of WT and mutant HsdR subunits. Agarose gel electrophoresis was used to analyse the single-site substrate pCFD30 that was incubated for 5 min at 37 °C with saturating amounts of MTase (20 nM) and HsdR (160 nM) in the presence of 4 mM ATP (Materials and Methods). CCC is covalently closed circular DNA (substrate), OC is open circle DNA (product cut in one strand) and FLL is full-length linear DNA (product cut in both strands). (b and c) Comparison of the cleavage time course for WT HsdR (b) and E165D HsdR (c) at 25 °C. pMDS27.3 and saturating MTase (40 nM) and HsdR (100 nM) were preincubated, and the reaction was initiated by the addition of ATP to 4 mM. Samples were removed at the time points indicated and immediately quenched. The DNA substrate and products were then separated by agarose gel electrophoresis and quantified by scintillation counting (Materials and Methods). The different relative mobilities of the FLL fragments reflect different running conditions for each gel (i.e., different values in V cm− 1). DNA dimers are a minor contaminant in our plasmid preparations. (d) Fitting of the CCC data from WT (b), E165D (c) and E165A (data not shown) using Eq. (1) as described in Materials and Methods. The fitted parameters are given in Table 2. Under our reaction conditions, DNA cleavage by EcoR124I never goes to completion because of a background inhibition activity that competes with the translocation/cleavage process. Regardless of the observed cleavage rates, very little change in the relative levels of the species is observed beyond ∼ 5–10 min. This has been noted previously with EcoR124I using both WT enzyme and QxxxY motif mutants8 and has also been observed with EcoKI, but not with EcoAI (F. Peske, unpublished observations). The effect appears to be dependent on changes to the DNA that make it resistant and is not dependent on inhibition of the protein. However, the exact nature of the inhibition of DNA cleavage is unclear and is currently still under investigation (F. Peske, unpublished observations).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602864&req=5

fig2: DNA cleavage activity of motif II and III nuclease mutants of EcoR124I. (a) Comparison of cleavage end points of WT and mutant HsdR subunits. Agarose gel electrophoresis was used to analyse the single-site substrate pCFD30 that was incubated for 5 min at 37 °C with saturating amounts of MTase (20 nM) and HsdR (160 nM) in the presence of 4 mM ATP (Materials and Methods). CCC is covalently closed circular DNA (substrate), OC is open circle DNA (product cut in one strand) and FLL is full-length linear DNA (product cut in both strands). (b and c) Comparison of the cleavage time course for WT HsdR (b) and E165D HsdR (c) at 25 °C. pMDS27.3 and saturating MTase (40 nM) and HsdR (100 nM) were preincubated, and the reaction was initiated by the addition of ATP to 4 mM. Samples were removed at the time points indicated and immediately quenched. The DNA substrate and products were then separated by agarose gel electrophoresis and quantified by scintillation counting (Materials and Methods). The different relative mobilities of the FLL fragments reflect different running conditions for each gel (i.e., different values in V cm− 1). DNA dimers are a minor contaminant in our plasmid preparations. (d) Fitting of the CCC data from WT (b), E165D (c) and E165A (data not shown) using Eq. (1) as described in Materials and Methods. The fitted parameters are given in Table 2. Under our reaction conditions, DNA cleavage by EcoR124I never goes to completion because of a background inhibition activity that competes with the translocation/cleavage process. Regardless of the observed cleavage rates, very little change in the relative levels of the species is observed beyond ∼ 5–10 min. This has been noted previously with EcoR124I using both WT enzyme and QxxxY motif mutants8 and has also been observed with EcoKI, but not with EcoAI (F. Peske, unpublished observations). The effect appears to be dependent on changes to the DNA that make it resistant and is not dependent on inhibition of the protein. However, the exact nature of the inhibition of DNA cleavage is unclear and is currently still under investigation (F. Peske, unpublished observations).

Mentions: To test the nuclease activity of the HsdR mutants in vitro, we measured DNA cleavage of single-site plasmids (Materials and Methods).8 We first tested cleavage activity after a fixed incubation time. For the WT HsdR, maximum cleavage (as judged by the production of ∼ 80% linear DNA) can be obtained using a > 4-fold molar excess of HsdR relative to the MTase–DNA concentration and a reaction time of ∼ 5 min.5 An excess of HsdR is required because of the dynamic nature of the HsdR interaction with the MTase.19 However, to ensure that we could capture any cleavage event that occurred with our mutant enzymes, we increased the molar excess of HsdR to a > 20-fold molar excess. No cleavage activity was observed over 5 min with the D151A, E165H or K167A mutant (Fig. 2a). Increasing the HsdR concentration or the incubation time did not alter these results (data not shown). Some partial DNA nicking and the production of a small amount of full-length linear DNA were observed with the E165A and E165D mutants. These results are completely consistent with the in vivo results (Table 1); a DNA nicking activity is insufficient to stop phage infection.30


The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I.

Sisáková E, Weiserová M, Dekker C, Seidel R, Szczelkun MD - J. Mol. Biol. (2008)

DNA cleavage activity of motif II and III nuclease mutants of EcoR124I. (a) Comparison of cleavage end points of WT and mutant HsdR subunits. Agarose gel electrophoresis was used to analyse the single-site substrate pCFD30 that was incubated for 5 min at 37 °C with saturating amounts of MTase (20 nM) and HsdR (160 nM) in the presence of 4 mM ATP (Materials and Methods). CCC is covalently closed circular DNA (substrate), OC is open circle DNA (product cut in one strand) and FLL is full-length linear DNA (product cut in both strands). (b and c) Comparison of the cleavage time course for WT HsdR (b) and E165D HsdR (c) at 25 °C. pMDS27.3 and saturating MTase (40 nM) and HsdR (100 nM) were preincubated, and the reaction was initiated by the addition of ATP to 4 mM. Samples were removed at the time points indicated and immediately quenched. The DNA substrate and products were then separated by agarose gel electrophoresis and quantified by scintillation counting (Materials and Methods). The different relative mobilities of the FLL fragments reflect different running conditions for each gel (i.e., different values in V cm− 1). DNA dimers are a minor contaminant in our plasmid preparations. (d) Fitting of the CCC data from WT (b), E165D (c) and E165A (data not shown) using Eq. (1) as described in Materials and Methods. The fitted parameters are given in Table 2. Under our reaction conditions, DNA cleavage by EcoR124I never goes to completion because of a background inhibition activity that competes with the translocation/cleavage process. Regardless of the observed cleavage rates, very little change in the relative levels of the species is observed beyond ∼ 5–10 min. This has been noted previously with EcoR124I using both WT enzyme and QxxxY motif mutants8 and has also been observed with EcoKI, but not with EcoAI (F. Peske, unpublished observations). The effect appears to be dependent on changes to the DNA that make it resistant and is not dependent on inhibition of the protein. However, the exact nature of the inhibition of DNA cleavage is unclear and is currently still under investigation (F. Peske, unpublished observations).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602864&req=5

fig2: DNA cleavage activity of motif II and III nuclease mutants of EcoR124I. (a) Comparison of cleavage end points of WT and mutant HsdR subunits. Agarose gel electrophoresis was used to analyse the single-site substrate pCFD30 that was incubated for 5 min at 37 °C with saturating amounts of MTase (20 nM) and HsdR (160 nM) in the presence of 4 mM ATP (Materials and Methods). CCC is covalently closed circular DNA (substrate), OC is open circle DNA (product cut in one strand) and FLL is full-length linear DNA (product cut in both strands). (b and c) Comparison of the cleavage time course for WT HsdR (b) and E165D HsdR (c) at 25 °C. pMDS27.3 and saturating MTase (40 nM) and HsdR (100 nM) were preincubated, and the reaction was initiated by the addition of ATP to 4 mM. Samples were removed at the time points indicated and immediately quenched. The DNA substrate and products were then separated by agarose gel electrophoresis and quantified by scintillation counting (Materials and Methods). The different relative mobilities of the FLL fragments reflect different running conditions for each gel (i.e., different values in V cm− 1). DNA dimers are a minor contaminant in our plasmid preparations. (d) Fitting of the CCC data from WT (b), E165D (c) and E165A (data not shown) using Eq. (1) as described in Materials and Methods. The fitted parameters are given in Table 2. Under our reaction conditions, DNA cleavage by EcoR124I never goes to completion because of a background inhibition activity that competes with the translocation/cleavage process. Regardless of the observed cleavage rates, very little change in the relative levels of the species is observed beyond ∼ 5–10 min. This has been noted previously with EcoR124I using both WT enzyme and QxxxY motif mutants8 and has also been observed with EcoKI, but not with EcoAI (F. Peske, unpublished observations). The effect appears to be dependent on changes to the DNA that make it resistant and is not dependent on inhibition of the protein. However, the exact nature of the inhibition of DNA cleavage is unclear and is currently still under investigation (F. Peske, unpublished observations).
Mentions: To test the nuclease activity of the HsdR mutants in vitro, we measured DNA cleavage of single-site plasmids (Materials and Methods).8 We first tested cleavage activity after a fixed incubation time. For the WT HsdR, maximum cleavage (as judged by the production of ∼ 80% linear DNA) can be obtained using a > 4-fold molar excess of HsdR relative to the MTase–DNA concentration and a reaction time of ∼ 5 min.5 An excess of HsdR is required because of the dynamic nature of the HsdR interaction with the MTase.19 However, to ensure that we could capture any cleavage event that occurred with our mutant enzymes, we increased the molar excess of HsdR to a > 20-fold molar excess. No cleavage activity was observed over 5 min with the D151A, E165H or K167A mutant (Fig. 2a). Increasing the HsdR concentration or the incubation time did not alter these results (data not shown). Some partial DNA nicking and the production of a small amount of full-length linear DNA were observed with the E165A and E165D mutants. These results are completely consistent with the in vivo results (Table 1); a DNA nicking activity is insufficient to stop phage infection.30

Bottom Line: We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain.In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics.The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

Show MeSH
Related in: MedlinePlus