Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Cellular Response to DNA Damage

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions repaired by non-homologous end joining (NHEJ) or homologous recombination (HR).
• RIF1 protein and its effector Shieldin complex are known negative regulators of DNA resection, a key step in HR.
• The MRN (MRE11-RAD50-NBS1) complex plays a role in sensing DSBs and initiating resection.

Purpose of the Study:

• To investigate the mechanisms by which RIF1 inhibits DNA resection beyond its known effector, Shieldin.
• To identify alternative RIF1 effectors involved in regulating the early DNA damage response.
• To elucidate the role of RIF1's effectors in preventing inappropriate homologous recombination (HR) activation.

Main Methods:

• Immunofluorescence microscopy to track protein localization at DSB sites.
• Analysis of CtIP accumulation and DNA resection levels in wild-type and mutant cells.
• Assessment of HR repair dynamics following DNA damage induction via irradiation.

Main Results:

• RIF1, but not Shieldin, inhibits the immediate accumulation of CtIP at DSB sites.
• Protein phosphatase 1 (PP1) localizes to DSB sites in a RIF1-dependent manner.
• PP1 suppresses CtIP accumulation and limits MRN complex-mediated resection, acting as a distinct RIF1 effector.
• Combined depletion of Shieldin and PP1 leads to elevated HR early after irradiation.

Conclusions:

• RIF1 utilizes protein phosphatase 1 (PP1) as a distinct effector, separate from Shieldin, to regulate DNA resection.
• PP1 acts in the early phase of the DNA damage response to inhibit resection before Shieldin's action.
• This RIF1-PP1 pathway prevents precocious homologous recombination (HR) activation, ensuring timely and accurate DNA repair.