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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.

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Bulk-solution measurement of DNA translocation and HsdR turnover by WT and mutant HsdR subunits using triplex displacement.19 Triplex displacement profiles were derived from stopped-flow experiments at a subsaturating HsdR concentration. Reactions were initiated by mixing preincubated triplex DNA (2093 bp between the EcoR124I and triplex binding sites), MTase and HsdR with an equal volume of reaction buffer plus ATP. The final solution contains 40 nM MTase, 1 nM HsdR, 5 nM DNA, 2.5 nM triplex and 4 mM ATP. Note that only 25% of the translocation events can lead to triplex displacement since there are 10 nM HsdR binding sites available (2 per DNA-bound MTase) but only 2.5 nM TFO bound.19 Experimental data are shown as scatter points, and continuous lines are fits to a single exponential.
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fig6: Bulk-solution measurement of DNA translocation and HsdR turnover by WT and mutant HsdR subunits using triplex displacement.19 Triplex displacement profiles were derived from stopped-flow experiments at a subsaturating HsdR concentration. Reactions were initiated by mixing preincubated triplex DNA (2093 bp between the EcoR124I and triplex binding sites), MTase and HsdR with an equal volume of reaction buffer plus ATP. The final solution contains 40 nM MTase, 1 nM HsdR, 5 nM DNA, 2.5 nM triplex and 4 mM ATP. Note that only 25% of the translocation events can lead to triplex displacement since there are 10 nM HsdR binding sites available (2 per DNA-bound MTase) but only 2.5 nM TFO bound.19 Experimental data are shown as scatter points, and continuous lines are fits to a single exponential.

Mentions: Since for each of the mutants there is the possibility of (1) stalling during initiation, (2) reduced translocation rates and (3) altered translocation lifetimes (Figs. 3 and 5; Table 2), it is likely that the dynamics of HsdR turnover is also altered. We tested this using the triplex displacement assay under conditions in which HsdR is subsaturating compared with the concentration of MTase–DNA complexes (Fig. 6).19 To get complete triplex displacement under these conditions, each HsdR must encounter, translocate and dissociate from multiple MTase–DNA complexes. The observed rate of triplex displacement then reflects the kinetics of each of these processes. Sample profiles are shown for each of the mutants on a linear DNA with a 2093-bp spacing between the triplex and EcoR124I recognition site. With the exception of E165D, all the mutants showed a slower displacement than the WT; consistent with the other assays, the E165H mutant was the slowest, while D151A, E165A and K167A were all similar.


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)

Bulk-solution measurement of DNA translocation and HsdR turnover by WT and mutant HsdR subunits using triplex displacement.19 Triplex displacement profiles were derived from stopped-flow experiments at a subsaturating HsdR concentration. Reactions were initiated by mixing preincubated triplex DNA (2093 bp between the EcoR124I and triplex binding sites), MTase and HsdR with an equal volume of reaction buffer plus ATP. The final solution contains 40 nM MTase, 1 nM HsdR, 5 nM DNA, 2.5 nM triplex and 4 mM ATP. Note that only 25% of the translocation events can lead to triplex displacement since there are 10 nM HsdR binding sites available (2 per DNA-bound MTase) but only 2.5 nM TFO bound.19 Experimental data are shown as scatter points, and continuous lines are fits to a single exponential.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2602864&req=5

fig6: Bulk-solution measurement of DNA translocation and HsdR turnover by WT and mutant HsdR subunits using triplex displacement.19 Triplex displacement profiles were derived from stopped-flow experiments at a subsaturating HsdR concentration. Reactions were initiated by mixing preincubated triplex DNA (2093 bp between the EcoR124I and triplex binding sites), MTase and HsdR with an equal volume of reaction buffer plus ATP. The final solution contains 40 nM MTase, 1 nM HsdR, 5 nM DNA, 2.5 nM triplex and 4 mM ATP. Note that only 25% of the translocation events can lead to triplex displacement since there are 10 nM HsdR binding sites available (2 per DNA-bound MTase) but only 2.5 nM TFO bound.19 Experimental data are shown as scatter points, and continuous lines are fits to a single exponential.
Mentions: Since for each of the mutants there is the possibility of (1) stalling during initiation, (2) reduced translocation rates and (3) altered translocation lifetimes (Figs. 3 and 5; Table 2), it is likely that the dynamics of HsdR turnover is also altered. We tested this using the triplex displacement assay under conditions in which HsdR is subsaturating compared with the concentration of MTase–DNA complexes (Fig. 6).19 To get complete triplex displacement under these conditions, each HsdR must encounter, translocate and dissociate from multiple MTase–DNA complexes. The observed rate of triplex displacement then reflects the kinetics of each of these processes. Sample profiles are shown for each of the mutants on a linear DNA with a 2093-bp spacing between the triplex and EcoR124I recognition site. With the exception of E165D, all the mutants showed a slower displacement than the WT; consistent with the other assays, the E165H mutant was the slowest, while D151A, E165A and K167A were all similar.

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