Bloom syndrome (BS) is a rare human genetic disease in which patients exhibit growth retardation, immunodeficiency, infertility, photosensitivity, and predisposition to cancer. The gene defective in BS has recently been cloned (named BLM) and was found to belong to an evolutionarily conserved helicase family, called RecQ. The recombinant BLM protein has been shown to possess helicase activity in vitro, suggesting that BS could be caused by a defect in a DNA metabolic reaction such as replication or repair. Interestingly, the BLM gene belongs to the helicase family, like the genes mutated in Werner Syndrome and Rothmund-Thomson syndrome (RTS). All three diseases have some common features, such as genetic instability and predisposition to cancer. But each disease has its own distinctive symptoms. For example, WS patients prematurely display many age-related features, including osteoporosis, atherosclerosis, diabetes and cataracts, which are not observed in BS or RTS. Also, WS individuals do not show immunodeficiency or photosensivity like BS patients. To help understand the molecular mechanism of these human diseases, we are isolating the protein complexes containing each gene product. To investigate the mechanism of BS, we isolated from human HeLa extracts three complexes containing the helicase BLM defective in BS. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity that requires the presence of BLM, so that complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIa and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We find that BLM complexes isolated from a FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair. We showed that BLAP75 is a component of all three BLM complexes from HeLa cells. Using siRNA knockdown techniques, we showed that BLAP75 is essential for BLM complex stability in vivo. Consistent with a role in BLM-mediated processes, BLAP75 co-localized with BLM in subnuclear foci in response to DNA damage, and its depletion impaired the recruitment of BLM to these foci. Depletion of BLAP75 by siRNA also resulted in deficient phosphorylation of BLM during mitosis, as well as defective cell proliferation. Moreover, cells depleted of BLAP75 displayed an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, BLAP75 is an essential component of the BLM-associated cellular machinery that maintains genome integrity. After our work was published, two other labs used genetic approaches to show that the yeast homolog of BLAP75, named RMI1, is also a component of RecQ helicase-Topo IIIa complex, and is required for maintaining genome stability. Thus, biochemistry in human and genetics in yeast have reached the same conclusion. Together, these data suggest that BLAP75/RMI1 and its homologs in various species have a conserved function in guarding the genome. It was shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase III (hTOPO III ), can process a toxic DNA intermediate generated in DNA repair reactions into a non-toxic product by a mechanism termed dissolution. In a collaboration with I. Hickson, G. Brown, and L. Lis labs, it was found that RMI1 (new name for BLAP75) can strongly promote the dissolution catalyzed by hTOPO III by recruiting this enzyme to the toxic intermediate. This study demonstrates that BLM, hTOPO III and BLAP75/RMI1 function as a molecular machine that maintains genome stability by efficiently processing the toxic intermediates generated during DNA repair. Identification of this machine and its biochemical activity should provide new means to screen drugs and could eventually contribute to the development of cancer therapies. We discovered a new component of the BLM complex---RMI2. RMI2 interacts with RMI1 (BLAP75) through two OB-fold domains similar to those in RPA. The resulting complex, named RMI, differs from RPA in that it lacks obvious DNA binding activity. Nevertheless, RMI stimulates the dissolution of a homologous recombination intermediate in vitro and is essential for the stability, localization, and function of the BLM complex in vivo. Notably, inactivation of RMI2 in chicken DT40 cells results in an increased level of sister-chromatid exchange (SCE)--the hallmark feature of Bloom syndrome cells. Epistasis analysis revealed that RMI2 and BLM suppress SCE within the same pathway. A point mutation in the OB-domain of RMI2 disrupts the association between BLM and the rest of the complex, and abrogates the ability of RMI2 to suppress elevated SCE. Our data suggest that multi-OB-fold complexes mediate two modes of BLM action: via RPA-mediated protein-DNA interaction and via RMI-mediated protein-protein interactions. We have since collaborated with J. Keck's group and solved the crystal structure of the RMI1-RMI2 core complex. The overall structure strongly resembles two-thirds of the trimerization core of the eukaryotic single-strand DNA-binding protein, Replication Protein A. Immunoprecipitation experiments with RMI2 variants confirm key interactions that stabilize the RMI core interface. Disruption of this interface leads to a dramatic increase in cellular sister chromatid exchange events similar to that seen in BLM-deficient cells. The RMI core interface is therefore crucial for BLM dissolvasome assembly and may have additional cellular roles as a docking hub for other proteins. We have Rif1 as a novel component of the BLM complex. We found that Rif1 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 CTD domain of bacterial RNA polymerase;and this domain preferentially binds fork and 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.
