53BP1 Regulates DSB Repair Using Rif1 to Control 5′ End Resection

Abstract

The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or non-homologous end-joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genomewide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1, limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage, and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.53BP1 can influence the type of DNA repair at DSBs (1) as seen in immunoglobulin gene rearrangements (2-4) and in the fusion of telomeres rendered dysfunctional through the removal of the shelterin protein TRF2 (5), where 53BP1 enhances the mobility of damaged telomeres, thus potentially promoting the chance of telomeretelomere encounters. In Brca1-deficient cells, 53BP1 enhances aberrant NHEJ events that create lethal radial chromosomes in response to poly(ADP-ribose) polymerase PARP1 inhibitors (PARPi) (6). In this setting, 53BP1 may favor NHEJ-mediated mis-rejoining by blocking the DSB resection needed for HDR (6, 7). 53BP1 was shown to impede 5' end resection at dysfunctional telomeres lacking all shelterin proteins and similarly, telomeres lacking only TRF2 show evidence of 53BP1-dependent protection from resection (5,8). Based on the finding that an allele of 53BP1 (53BP1 28A ) lacking all potential ATM/ATR kinase S/TQ target sites did not support immunoglobulin Class Switch Recombination (CSR) and failed to generate radial chromosomes in Brca1-deficient cells (7), it appears that these functions of 53BP1 involve interacting partner(s) modulated by the S/TQ sites. One candidate 53BP1-interacting factor is Rif1, which localizes to DSBs and dysfunctional telomeres, in a manner that is dependent on ATM signaling (9-11). Rif1 was originally identified as part of the telomeric complex in budding yeast (12) and was recently shown to inhibit resection at yeast telomeres (13,14). In contrast, mammalian Rif1 has no known function at functional telomeres but contributes to the intra-S phase checkpoint, facilitates recovery from replication stress, and affects replication timing (10,(15)(16)(17) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe introduced 53BP1 28A and other 53BP1 mutant alleles (7) into immortalized TRF2 F/-53BP1 -/-mouse embryo fibroblasts (MEFs) and induced telomere dysfunction by deletion of TRF2 (Fig. 1A,B). The results showed that the S/TQ sites were required for the accumulation of Rif1 at deprotected telomeres, whereas the GAR, BRCT, and oligomerization domains of 53BP1 were not ( Fig. 1A-C; fig. S1). The functional significance of the Rif1-53BP1 interaction was addressed using a tel...

Rif1 and Exo1 regulate the genomic instability following telomere losses

“…Similarly, elevated levels of Rif1 were found in breast cancer and teratocarcinomas (Wang et al ., 2009; Li et al ., 2015). Since mammalian Rif1 participates in suppressing the HDR repair pathway in the G1 phase (Chapman et al ., 2013; Di Virgilio et al ., 2013; Escribano‐Díaz et al ., 2013; Zimmermann et al ., 2013), it was suggested that too much Rif1 drives illicit and error‐prone DSB repair, which alters the genome. However, our study shows that Rif1 can drive genomic instability in the absence of DSB repair.…”

CtIP-mediated resection is essential for viability and can operate independently of BRCA1

“…It was shown that 53BP1 inhibits resection, but that BRCA1 antagonizes 53BP1, allowing the nucleolytic processing of DNA ends in S phase (Bunting et al, 2010;Chapman et al, 2012). Furthermore PTIP and RIF1, both of which act downstream of 53BP1, inhibit BRCA1-associated DNA metabolism (Callen et al, 2013;Chapman et al, 2013;EscribanoDíaz et al, 2013;Feng et al, 2013;Zimmermann et al, 2013). Nevertheless, the mediators of resection in BRCA1/53BP1, BRCA1/PTIP, and BRCA1/RIF double-deficient cells have not been identified.…”

“…These conclusions were based on the findings that BRCA1-deficient cells exhibit a mild decrease in IR-induced RPA focus formation (Chen et al, 2008;Escribano-Díaz et al, 2013), that BRCA1 and CtIP inhibit RIF1 end-blocking activity in S/G2 (Escribano-Díaz et al, 2013;Zimmermann et al, 2013), and that cells expressing CtIP-327A are defective in HR (Yun and Hiom, 2009). However, subsequent studies in chicken (Nakamura et al, 2010), frog (Peterson et al, 2011), and mouse cells ; analogous to our CtIPnull cells reconstituted with CtIP-S327A), and direct measurements of gene conversion (Chandramouly et al, 2013), argue that the CtIP-BRCA1 interaction is dispensable for HR.…”

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

“…The increase in HR events in cells lacking 53BP1 and RAP80/BRCA1 foci implies that inhibiting their recruitment to foci has supportive effects on HR, consistent with recent findings identifying a complex regulatory interplay between the BRCA1/RAP80 complex and 53BP1 at the level of restricting DNA end resection at sites of DSBs. [40][41][42][43][44][45][46][47] …”