Rif1 Is Required for Resolution of Ultrafine DNA Bridges in Anaphase to Ensure Genomic Stability

Abstract

Sister-chromatid disjunction in anaphase requires the resolution of DNA catenanes by topoisomerase II together with Plk1-interacting checkpoint helicase (PICH) and Bloom's helicase (BLM). We here identify Rif1 as a factor involved in the resolution of DNA catenanes that are visible as ultrafine DNA bridges (UFBs) in anaphase to which PICH and BLM localize. Rif1, which during interphase functions downstream of 53BP1 in DNA repair, is recruited to UFBs in a PICH-dependent fashion, but independently of 53BP1 or BLM. Similar to PICH and BLM, Rif1 promotes the resolution of UFBs: its depletion increases the frequency of nucleoplasmic bridges and RPA70-positive UFBs in late anaphase. Moreover, in the absence of Rif1, PICH, or BLM, more nuclear bodies with damaged DNA arise in ensuing G1 cells, when chromosome decatenation is impaired. Our data reveal a thus far unrecognized function for Rif1 in the resolution of UFBs during anaphase to protect genomic integrity.

Knotty Problems during Mitosis: Mechanistic Insight into the Processing of Ultrafine DNA Bridges in Anaphase

“…Consistent with this, PICH possesses ATP-dependent dsDNA translocase activity (Biebricher et al 2013). In addition to the SNF2 region, PICH has accessory domains, including the PICH-family domain, and two TPR motifs reported to be involved in protein interactions (Hengeveld et al 2015;Pitchai et al 2017). PICH appears to have a high affinity for stretched dsDNA, which is consistent with the idea that UFBs must be under considerable tension created by the mitotic spindle (Baumann et al 2007;Biebricher et al 2013).…”

“…PICH is excluded from the nucleus during interphase and is only recruited to chromatin after nuclear envelope breakdown, whereupon it accumulates at centromeric loci. PICH seems to be the main recognition and recruitment factor for UFBs, as several other UFB-processing factors fail to localize to UFBs in the absence of PICH, such as members of the Bloom syndrome complex (Chan et al 2007) and RIF1 (Hengeveld et al 2015). This makes it difficult to detect or analyze UFBs in the absence of PICH.…”

A new class of ultrafine anaphase bridges generated by homologous recombination

“…RIF1 (Rapl-interacting factor 1) is another protein that is recruited by PICH to C-UFBs [56]. RIF1 plays multiple functions in different phases of the cell cycle.…”

“…RIF1 colocalizes with replication forks mostly at pericentromeric heterochromatin in mid-S phase and is required for the regulation of replication timing and the assembly of newly replicated heterochromatin [60,61]. Although RIF1 interacts directly with BLM [62], the localization of RIF1 on C-UFBs does not depend on BLM, and vice versa [56]. Depletion of RIF1 increases the formation of micronuclei and G1-phase 53BP1 nuclear bodies in response to ICRF-193 treatment, suggesting that RIF1 is required for the timely resolution of C-UFBs.…”

“…In telophase, most of the HR-UFBs are exclusively coated with RPA, indicating that duplex DNA bridges are converted to ssDNA. Furthermore, RPA binding to HR-UFBs and C-UFBs are dependent on PICH/BLM [22,56]. These results lead us to propose a common mechanism for the processing of HR-UFBs, FS-UFBs and C-UFBs in late anaphase/telophase: PICH recruits BLM to unwind dsDNA present in the UFBs to generate single-stranded bridges that are coated with RPA [22].…”