Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity

Area of Science:

• Biochemistry
• Molecular Biology
• DNA Repair

Background:

• Human PMS2 (hPMS2) homologues are crucial for DNA mismatch repair, performing nicks near misincorporated nucleotides.
• These homologues' endonuclease activity is stimulated by Mn(++) ions.
• While ATP is required for hPMS2 and yPMS1, it inhibits homologous bacterial MutL proteins, creating a research gap.

Purpose of the Study:

• To investigate the effect of ATP on the Mn(++)-induced endonuclease activity of Aquifex aeolicus MutL (Aae MutL).
• To identify key functional domains and motifs within Aae MutL responsible for its nicking activity.

Main Methods:

• Enzyme kinetic assays were performed using supercoiled pBR322 DNA with full-length and mutant Aae MutL proteins.
• Experiments involved single time point, enzyme titration, and reaction time course analyses.
• Mutational analysis focused on conserved motifs like DQHA(X)(2)E(X)(4)E and CPHGRP, and a C-terminal truncation was also tested.

Main Results:

• ATP significantly stimulated the Mn(++)-induced nicking activity of Aae MutL, increasing the maximum velocity.
• AMPPNP (a non-hydrolyzable ATP analog) also stimulated activity, suggesting ATP hydrolysis is not required.
• A truncated Aae MutL protein (123 C-terminal residues) retained nicking activity, while mutations in the DQHA(X)(2)E(X)(4)E and CPHGRP motifs abolished activity.

Conclusions:

• ATP enhances the Mn(++)-dependent endonuclease activity of Aae MutL, independent of ATP hydrolysis.
• The C-terminal 123 amino acids of Aae MutL are sufficient for its nicking function.
• Conserved motifs, particularly DQHA(X)(2)E(X)(4)E and CPHGRP, are essential for Aae MutL's endonucleolytic function.