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Related Experiment Video

Updated: Jan 23, 2026

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Published on: November 1, 2019

DNA duplex recognition activates Exo1 nuclease activity.

Yuxi Li^1,2, Jiangchuan Shen¹, Hengyao Niu³

¹Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405.

The Journal of Biological Chemistry

|June 12, 2019

Summary

DNA hairpins activate Exonuclease 1 (Exo1) nuclease activity. A specific residue, Lysine 185, is crucial for Exo1

Keywords:

DNA binding protein DNA end resection DNA endonuclease DNA hairpin DNA repair DNA structure Exo1 homologous recombination replication protein A (RPA)

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Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Published on: November 1, 2012

Area of Science:

Genetics
Molecular Biology
Biochemistry

Background:

Exonuclease 1 (Exo1) is a conserved eukaryotic nuclease vital for genome stability.
Its activation mechanism, crucial for DNA repair and replication, remains incompletely understood.
Previous studies showed Exo1 digests single-stranded DNA (ssDNA) but not specific oligonucleotides.

Purpose of the Study:

To elucidate the activation mechanism of Exonuclease 1 (Exo1).
To identify the structural features of DNA that trigger Exo1 nuclease activity.
To investigate the role of specific Exo1 residues in DNA substrate recognition and activation.

Main Methods:

Purification of recombinant Exonuclease 1 (Exo1).
Nuclease assays to measure Exo1 activity on various DNA substrates.
Electrophoretic mobility shift assays (EMSAs) to study DNA-protein interactions.
Site-directed mutagenesis to assess the function of specific Exo1 residues (e.g., Lys185).

Main Results:

DNA hairpins with stem sizes of 4 base pairs or longer activate Exo1-mediated ssDNA digestion.
Exo1 utilizes the evolutionarily conserved residue Lysine 185 (Lys185) for duplex DNA recognition.
Lys185 interacts with the phosphate backbone of the DNA, facilitating activation on both ssDNA and dsDNA.
Mutating Lys185 to Alanine (exo1-K185A) impaired duplex DNA digestion, partially rescued by longer DNA overhangs.
Replication protein A (RPA) inhibits Exo1 activity on longer overhangs, likely by displacing Exo1 from ssDNA.

Conclusions:

Exo1 activation is generally controlled by contact with duplex DNA structures.
This duplex recognition mechanism is particularly important for Exo1's function on duplex DNA with RPA-bound ssDNA.
Lys185 is a key residue for Exo1's ability to recognize and be activated by duplex DNA structures.