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Related Experiment Video

Updated: Jul 9, 2026

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

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Published on: October 14, 2025

Basic-patch mutations in bacteriophage T4 Rad50 uncouple ATPase activation from processive nuclease activity.

Tibebe A Teklemariam¹, Ryan M Finnerty², Jennifer B Coats³

¹Washington University School of Medicine in St Louis Continuing Medical Education, United States.

Bioscience Reports

|July 8, 2026

Summary

Mutations in a basic patch on Rad50 reveal an allosteric pathway. This pathway links DNA binding to ATP hydrolysis and nuclease activity in the Mre11/Rad50 (MR) complex, crucial for DNA repair.

Keywords:

ATPase activity Allostery Bacteriophage T4 Mre11 Nuclease activity Rad50

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Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

Published on: May 24, 2017

Area of Science:

Biochemistry
Molecular Biology
DNA Repair

Background:

The Mre11/Rad50 (MR) complex is essential for DNA double-strand break repair.
The complex utilizes ATP binding and hydrolysis for DNA processing.
The precise mechanism of Rad50 ATPase activation by Mre11 and DNA is not fully understood.

Purpose of the Study:

Investigate the role of a basic patch on T4 Rad50 (Arg154, Arg155, Lys156) in DNA binding and ATPase activity.
Elucidate the allosteric mechanisms governing MR complex function.

Main Methods:

Site-directed mutagenesis of T4 Rad50 residues.
Enzyme kinetics assays to measure ATPase activity.
DNA binding affinity measurements.
Assays for nucleotide excision activity.

Main Results:

Mutations in the basic patch showed modest effects on DNA binding but significantly altered ATPase activity.
Mutants exhibited enhanced ATPase activation with Mre11 alone, suggesting a shift towards an active state.
Despite increased ATPase activity, ATP-dependent nuclease activity was impaired, particularly in the TripleA mutant.
A mutation at Asp479 indicated long-range coupling within the complex.

Conclusions:

The basic patch on Rad50 is not a primary DNA-contact surface but participates in an allosteric pathway.
This pathway connects Mre11 and DNA engagement to productive ATP hydrolysis.
Efficient nuclease activity requires more than just enhanced ATP hydrolysis; coupling to DNA processing is critical.