Dynamics of RIF1 SUMOylation is regulated by PIAS4 in the maintenance of Genomic Stability

Ramesh Kumar,Chit Fang Cheok

doi:10.1038/s41598-017-16934-w

Abstract

RIF1 plays a key role in inhibiting DNA end resection and promoting NHEJ mediated DNA double stand break repair in G1. However, whether SUMOlyation may regulate RIF1 functions is still largely unknown. Here, we report that RIF1 is SUMOlyated in response to DNA damage. We identified PIAS4 as the primary SUMO E3 ligase required for the SUMOylation of RIF1 protein. Mammalian cells compromised of PIAS4 expression, show impaired RIF1 SUMOylation and defective for the disassembly of DNA damage responsive RIF1 foci. Mechanistically, we show that PIAS4 knockdown abrogates UHRF1-dependent ubiquitination of RIF1, compromising RIF1 protein turnover. We detected intense RPA foci that colocalize with RIF1 foci in PIAS4 knockdown cells. These data highlight an important role of PIAS4-dependent regulation of RIF1, likely mediated by SUMOylation, in the disassembly of RIF1 DNA damage response (DDR) foci. We propose that unresolved RIF1 protein at sites of DNA damage in PIAS4-depleted cells largely accumulates in S phase, and subsequently leads to DNA double strand breaks. Therefore, PIAS4 promotes genomic stability by regulating the timely removal of RIF1 from sites of DNA damage.

Highlights

RIF1 plays a key role in inhibiting DNA end resection and promoting NHEJ mediated DNA double stand break repair in G1
RIF1 SUMOylation is increased in response to DNA double strand breaks
Coordinated protein SUMOylation and ubiquitination are key PTMs required for the recruitment and repair of double strand breaks (DSBs) in a timely manner[11,50,51]

Summary

Introduction

RIF1 plays a key role in inhibiting DNA end resection and promoting NHEJ mediated DNA double stand break repair in G1. Mammalian cells compromised of PIAS4 expression, show impaired RIF1 SUMOylation and defective for the disassembly of DNA damage responsive RIF1 foci. We detected intense RPA foci that colocalize with RIF1 foci in PIAS4 knockdown cells These data highlight an important role of PIAS4-dependent regulation of RIF1, likely mediated by SUMOylation, in the disassembly of RIF1 DNA damage response (DDR) foci. We propose that unresolved RIF1 protein at sites of DNA damage in PIAS4-depleted cells largely accumulates in S phase, and subsequently leads to DNA double strand breaks. Coordinated PIAS1 and PIAS4 mediated protein SUMOylation and ubiquitination facilitate the distribution of DDR components (MDC1, BRCA1 and 53BP1) at the sites of DNA breaks and promote the repair process[12]. Earlier studies have revealed a function for SUMO and ubiquitin in the recruitment and disassembly of DNA repair foci to prevent genomic instability[19,20,21,22]

Methods

Results

Conclusion