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Updated: Apr 25, 2026
Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins
Published on: February 25, 2017
Catalytic mechanism of bacteriophage T4 Rad50 ATP hydrolysis
Timothy J Herdendorf1, Scott W Nelson
1Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.
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.
Insights
This study investigates the ATP hydrolysis mechanism of T4 Rad50 (gp46), revealing that chemistry is rate-limiting. The findings suggest an asymmetric, dissociative-like transition state and highlight allosteric communication between Rad50 active sites.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair Mechanisms
Background:
- Spontaneous double-strand breaks (DSBs) are critical DNA lesions.
- Mre11-Rad50 (MR) complex is essential for initial DSB processing.
- Understanding the catalytic mechanism of Rad50 ATPase is key to DNA repair.
Purpose of the Study:
- To elucidate the kinetic and catalytic mechanism of T4 Rad50 (gp46) ATP hydrolysis.
- To investigate the role of Mre11 (gp47) and DNA in Rad50's ATPase activity.
- To explore the transition state and allosteric regulation of the MR complex.
Main Methods:
- Single-turnover and pre-steady state kinetic analyses.
- pH-rate profiles and deuterium solvent isotope effect measurements.
- Site-directed mutagenesis of Walker A/B and H-loop residues.
Main Results:
- The rate-limiting step for Rad50, MR, and MR-DNA complexes involves chemistry or pre-catalytic conformational change.
- DNA binding does not alter the rate-limiting step.
- Evidence suggests an asymmetric, dissociative-like transition state for ATP hydrolysis.
- Mutations impact allosteric communication between Rad50 active sites.
Conclusions:
- ATP hydrolysis by T4 Rad50 proceeds via an asymmetric, dissociative-like transition state.
- Allosteric communication between Rad50 active sites is crucial for cooperativity.
- These findings provide insights into the DNA repair machinery's function.
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