Lesion recognition and cleavage by endonuclease V: a single-molecule study

Area of Science:

• Molecular Biology
• Enzymology
• Biochemistry

Background:

• Endonuclease V (endo V) is crucial for DNA repair, specifically targeting and cleaving deoxyinosine in deaminated DNA.
• Enzymatic activity is modulated by divalent metal ions, with Ca2+ linked to protein binding and Mg2+ to DNA cleavage.

Purpose of the Study:

• To investigate the single-molecule kinetics of endo V during its catalytic cycle.
• To elucidate the distinct roles of Ca2+ and Mg2+ in endo V enzymatic activity using a deoxyinosine-containing substrate.

Main Methods:

• Utilized single-molecule fluorescence resonance energy transfer (FRET) to monitor endo V activity.
• Labeled single-stranded DNA (ssDNA) with TAMRA (donor) and endo V with Cy5 (acceptor).
• Measured time lapses of FRET signals corresponding to substrate association, recognition, and dissociation.

Main Results:

• In the presence of Mg2+, association, recognition, and dissociation kinetics were measured at 5.9s, 14.5s, and 9.1s, respectively.
• Deoxyinosine recognition kinetics were minimally affected by the type of metal ion.
• The combination of Ca2+ and Mg2+ prolonged association and dissociation events compared to Mg2+ alone.

Conclusions:

• The findings support a model where endo V possesses two distinct metal binding sites.
• These sites differentially regulate enzymatic activities, with Ca2+ influencing protein interactions and Mg2+ facilitating DNA cleavage.
• This provides mechanistic insights into metal ion-dependent DNA repair pathways.