An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

Rosa, Metzere Bierlein De la; Nelson, Scott W.

doi:10.1074/jbc.m111.256305

Cited by 27 publications

(31 citation statements)

References 36 publications

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“…We have recently characterized the WT bacteriophage T4 MR-D complex, 18 as well as residues in the signature motif, 17 the D-loop aspartate, and Walker A asparagine. 24 In this study, the remaining motif residues have been mutated and ATPase kinetic characterization has been performed.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Interestingly, three of the mutants (N38A, S43A, and D512A) that were significantly affected (∼50-fold decrease in activity) are all associated with the Walker A-motif and similar in magnitude to the diminution of the only other Walker A mutation (31-fold for K42M). 18,24 Determination of the ATPase Reaction Rate-Limiting Step for Rad50, MR, and the MR-D Complex. The Rad50 ATPase reaction was followed under single-turnover conditions using a quenched-flow apparatus.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Herdendorf

Nelson

2014

Biochemistry

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Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH-rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites. ABSTRACT: Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH−rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites.DNA double-strand breaks (DSBs) are one of the most harmful types of DNA damage. 1 In humans, DSBs occur, on average, 50 times per cell cycle 2 and arise during normal physiological processes. Examples of these processes include free radical production, class-switch recombination for antibody effector modulation, 3,4 Spo11-dependent DSB during meiosis, 5 and the collapse of a stalled replication fork during DNA re...

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Section: ■ Discussionmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Herdendorf

Nelson

2014

Biochemistry

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“…In contrast, ATP reduces the nuclease activity of MR 2CS , MR 2KP , MR xlink as compared with reactions in the absence of ATP. This behavior is often observed in mutant MR complexes with low ATPase activity or in the presence of nonhydrolyzable ATP (15,44,45). The inhibition may be due to the inability to dissociate from or translocate forward on the DNA substrate (i.e.…”

Section: Analysis Of Cxxc Mutants In Vivo-mentioning

confidence: 99%

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

Barfoot

Herdendorf

Behning

et al. 2015

Journal of Biological Chemistry

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Background: The Mre11-Rad50 (MR) complex coordinates DNA double strand break repair. Results: Mutations of the Rad50 coiled-coil disrupt the enzymatic functions of the MR complex. Conclusion: The Rad50 coiled-coil regulates and mediates communication between the enzymatic domains of Rad50 and Mre11. Significance: The Rad50 coiled-coil plays a more direct role in the enzymatic function of Rad50 and Mre11 than was previously assumed.

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“…Because C pep -Mre11 is too small to be directly detectable via gel filtration analysis, we performed a simple competition assay. Previous pulldown experiments indicate tight binding and slow dissociation of MR complex (41). If C pepMre11 binds strongly to the same site on Rad50 as does WTMre11, then the MR complex should not form in the presence of C pep -Mre11.…”

Section: Steady-state Kinetics Of the Nuclease Activity For Wt Andmentioning

confidence: 99%

Autoinhibition of Bacteriophage T4 Mre11 by Its C-terminal Domain

Gao

Nelson

2014

Journal of Biological Chemistry

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Background: How nuclease activity of Mre11 is controlled by Rad50 is poorly understood. Results: Removal of the C-terminal domain of T4 Mre11 enhances its nuclease activity. Conclusion: The C terminus of Mre11 is autoinhibitory, and complex formation with Rad50 relieves this inhibition. Significance: This autoinhibition provides a mechanism for restricting the nuclease activity of Mre11 in the absence of Rad50.

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An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

Cited by 27 publications

References 36 publications

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

Autoinhibition of Bacteriophage T4 Mre11 by Its C-terminal Domain

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