Rif1 provides a new DNA-binding interface for the Bloom syndrome complex to maintain normal replication

Abstract

BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C-terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM-deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA-binding domain that resembles the aCTD domain of bacterial RNA polymerase a; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA-binding interface for the BLM complex to restart stalled replication forks.

A new class of ultrafine anaphase bridges generated by homologous recombination

“…These results indicate that RIF1 mainly localizes on double-stranded UFBs before they are converted to ssDNA by BLM. This is consistent with biochemical studies of RIF1 showing that its C-terminal region preferentially binds DNA forks and HJs compared with ssDNA [62]. However, the exact role of RIF1 in processing UFBs remains unclear.…”

“…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.…”

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

“…4). These constructs were designed to remove putative functional elements, including the C-terminal Dbf4-interacting region identified above, the HEAT repeats (Xu et al 2010;Sreesankar et al 2012), potential PP1 interaction motifs in the N-terminal domain (Sreesankar et al 2012), and a region with weak homology with a putative Holliday junction (HJ)-binding domain in vertebrate Rif1 (Xu et al 2010).…”

Structural and Biophysical Characterization of Murine Rif1 C Terminus Reveals High Specificity for DNA Cruciform Structures

“…It was reported that Rif1 requires its N terminus to accumulate at DSB sites (11), whereas the C terminus is required for interaction with BLM helicase (18). The N terminus of Rif1 is predicted to form 21 HEAT-type ␣-helical repeats (5,18), whereas the C-terminal region comprises three smaller parts: CRI, CRII, and CRIII (see Fig.…”

“…There are two conserved regions (CR) in the murine Rif1 (2418 amino acids): an N-terminal ϳ1000-amino acid region and a C-terminal ϳ350-amino acid region (5,18 ate protein-protein and protein-nucleic acid interactions, although functional mapping of domains or regions is limited. It was reported that Rif1 requires its N terminus to accumulate at DSB sites (11), whereas the C terminus is required for interaction with BLM helicase (18).…”