Abstract 2128: A new protein complex containing the Fanconi protein FANCM and Rif1

Abstract

Abstract Fanconi anemia (FA) is a genetic disease featuring characteristic developmental abnormalities, genomic instability and predisposition to cancer. The FA pathway consists of a “core complex,” including at least twelve proteins required for the monoubiquitylation of the FANCD2/FANCI protein complex and for other functions. Components downstream of the FANCD2/FANCI complex include breast cancer susceptibility components FANCD1/BRCA2, the partner of BRCA2 (Palb2/FANCN), and a helicase associated with BRCA1 (FANCJ/BACH1), and newly identified proteins including FAN1, FANCO/RAD51C, FANCP/SLX4. Together, the FA/BRCA pathway responds to replication stress and specific types of DNA damage such as DNA interstrand crosslinks. There are components in the pathway yet to be discovered, and the function of most of the FA proteins is unclear. Our lab uses assays in human cells and Xenopus cell-free assays to identify proteins that participate in the function of the FA pathway, and to analyze the function of the FA proteins in context with DNA replication. Xenopus extracts are a concentrated source of cell-cycle synchronized proteins that have been used for biochemical isolation and characterization of novel protein complexes. Because egg extracts contain stockpiles of nuclear proteins and are precisely synchronized in S or M phase, we reasoned that FA complexes isolated from egg extracts might be enriched for FA-associated proteins that could be difficult to detect in human cells. Using an antibody specific for the Xenopus ortholog of FANCM, we immunoisolated FANCM-containing complexes from egg extracts and analyzed the components by mass spectrometry. One FANCM subcomplex contained components of the FA core complex as expected, and a separate FANCM-complex contained the Xenopus ortholog of the DNA damage response protein, Rif1. Using assays in human cells, we found that Rif1 was undetectable in cells lacking FANCM but readily detectable in wild-type cells or in cells defective for downstream FA proteins. Rif1 co-precipitates with FANCM in wild-type cells, but is not detected in immune complexes in cell lines lacking FANCM, although Rif1 is expressed. These data suggest that FANCM and Rif1 are together in a complex in human cells, and that Rif1 or the Rif1/FANCM complex may be unstable when FANCM is absent or defective. Rif1 forms DNA damage induced nuclear foci that are reduced in cells depleted of FANCM, suggesting that FANCM is required for localization of Rif1 in subnuclear foci, perhaps at sites of DNA repair. We identified xRif1 as an interactor of xFANCM using a proteomics approach. Our data suggest that xFANCM is in two separate complexes, one containing FA proteins and the other containing Rif1. Rif1 was recently identified as a component of the BLM helicase complex, and was shown to promote recovery of stalled replication forks, raising the possibility that Rif1 may function with xFANCM during DNA damage response to maintain genomic stability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2128. doi:1538-7445.AM2012-2128