Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Rif1 protein plays a critical role in maintaining telomere stability, regulating DNA replication timing, and directing DNA double-strand break (DSB) repair pathway selection across various species.
• The precise molecular mechanisms underlying Rif1's diverse functions, particularly in DNA repair, are not fully understood.

Purpose of the Study:

• To investigate the posttranslational modifications of Rif1 and elucidate their role in DNA repair.
• To identify the specific modification and the enzymes responsible for it.
• To determine how this modification impacts Rif1's function at DSBs and influences repair pathway choice.

Main Methods:

• Utilized biochemical assays to identify S-acylation of Rif1 at specific cysteine residues (C466 and C473) within its N-terminal domain.
• Investigated the role of the DHHC family palmitoyl acyltransferase Pfa4 in Rif1 S-acylation.
• Assessed the impact of Rif1 S-acylation on DSB accumulation, DNA end-resection, and the choice between non-homologous end-joining (NHEJ) and homologous recombination repair pathways.

Main Results:

• Demonstrated that Rif1 is modified by S-acylation at cysteine residues C466 and C473, catalyzed by the enzyme Pfa4.
• Showed that S-acylation of Rif1 enhances its recruitment to DSBs, leading to reduced DNA end-resection.
• Found that S-acylated Rif1 promotes DSB repair via the NHEJ pathway and localizes to the inner nuclear membrane, influencing compartmentalized repair.

Conclusions:

• Identified S-acylation as a novel posttranslational modification regulating the function of Rif1 in DNA repair.
• Revealed that S-acylation of Rif1 is essential for efficient DSB repair by promoting NHEJ and attenuating DNA end-resection.
• Established a mechanism where S-acylated Rif1 contributes to compartmentalized DSB repair pathway choice by sequestration at the inner nuclear membrane.