Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1...

Area of Science:

• Molecular Biology
• DNA Repair
• Cell Cycle Regulation

Background:

• Yeast Rif2 protein inhibits Mre11 nuclease and Tel1 kinase activation via its MIN motif.
• Rif2 binds Rad50 and stimulates its ATPase activity, but the precise mechanism of Tel1 inhibition at DNA double-strand breaks (DSBs) is unclear.

Purpose of the Study:

• To elucidate the mechanism of Rif2-mediated Tel1 inhibition at DSBs.
• To characterize the role of Rif2 in regulating Tel1 recruitment and activity.

Main Methods:

• AlphaFold-Multimer modelling to identify the Rif2 MIN-Rad50 interaction surface.
• Engineering of the rif2-S6E mutation to enhance Rif2-Rad50 interaction.
• Analysis of hairpin cleavage, Tel1 activation, and MRX complex dynamics.

Main Results:

• The rif2-S6E mutation enhances Rif2-Rad50 interaction, impairs hairpin cleavage, and diminishes Tel1 activation.
• Rif2 directly inhibits Tel1 recruitment to DSBs by promoting an ADP-bound MRX conformation.
• Rif2 binding to Rad50 reduces Tel1-MRX interaction and impairs DSB end-tethering.

Conclusions:

• Rif2 acts as a direct inhibitor of Tel1 recruitment to DSBs.
• Tel1 plays a crucial role in the bridging of DSB ends, a function regulated by Rif2.
• The study reveals a novel mechanism for controlling Tel1 activity in DNA repair.