Singh TR, Ali AM, Busygina V, Raynard S, Fan Q, Du CH et al.BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome. Genes Dev 22:2856-2868

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA.
Genes & Development (Impact Factor: 10.8). 11/2008; 22(20):2856-68. DOI: 10.1101/gad.1725108
Source: PubMed


Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIalpha and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo IIIalpha-BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIalpha and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.

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    • "This intermediate is unlinked by Topoisomerase IIIα (TopoIIIα) to generate non-crossover products [6]. BLM and TopoIIIα work in the context of a hetero-complex named the dissolvasome [10] that also includes the “RecQ-mediated instability factors”, RMI1 and RMI2 [11,12]. The interaction and concerted activities of the BLM and TopoIIIα complex is sufficient to drive dHJ dissolution [6] but RMI1 has been found to stimulate this reaction [13,14]. "
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    ABSTRACT: Processing of homologous recombination intermediates is tightly coordinated to ensure that chromosomal integrity is maintained and tumorigenesis avoided. Decatenation of double Holliday junctions, for example, is catalysed by two enzymes that work in tight coordination and belong to the same 'dissolvasome' complex. Within the dissolvasome, the RecQ-like BLM helicase provides the translocase function for Holliday junction migration, while the topoisomerase III alpha-RMI1 subcomplex works as a proficient DNA decatenase, together resulting in double-Holliday-junction unlinking. Here, we review the available architectural and biochemical knowledge on the dissolvasome machinery, with a focus on the structural interplay between its components.
    Cell and Bioscience 07/2014; 4(1):36. DOI:10.1186/2045-3701-4-36 · 3.63 Impact Factor
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    • "The primers used in complementary DNA (cDNA) subcloning and mutant generation are listed in Supplementary Table S2. FANCL cDNA was amplified by polymerase chain reaction (PCR) from pDEST20-FANCL (15) with primers FL1 and FL2 and was inserted between the EcoRI and SalI sites in pMAL-TEV (17) to yield pMAL-TEV–FANCL. The FANCL W212A/L214A mutant was constructed by replacing the ClaI–SalI fragment of pMAL-TEV–FANCL with an FANCL PCR product (using FL3 and FL2 primers) that harbors the double mutation. "
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    ABSTRACT: FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.
    Nucleic Acids Research 03/2014; 42(9). DOI:10.1093/nar/gku198 · 9.11 Impact Factor
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    • "Comparable to mutants of the respective RecQ helicase involved in the RTR complex, the loss of RMI1 also leads to an elevated frequency of recombination events (Chang et al., 2005; Yin et al., 2005). RMI2 was identified as a fourth RTR complex partner in mammals and is also required for the assembly of the RTR complex and the stimulation of dissolution (Singh et al., 2008; Xu et al., 2008). The RTR complex is additionally involved in further, early steps of recombination, such as end resection, which could be shown in yeast (Zhu et al., 2008). "
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    ABSTRACT: The RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RMI1 is involved in the processing of DNA recombination intermediates in all eukaryotes. In Arabidopsis thaliana the complex partners RECQ4A, topoisomerase 3α and RMI1 have been shown to be involved in DNA repair and in the suppression of homologous recombination in somatic cells. Interestingly, mutants of AtTOP3A and AtRMI1 are also sterile due to extensive chromosome breakage in meiosis I, a phenotype that seems to be specific for plants. Although both proteins are essential for meiotic recombination it is still elusive on what kind of intermediates they are acting on. Recent data indicate that the pattern of non-crossover (NCO)-associated meiotic gene conversion (GC) differs between plants and other eukaryotes, as less NCOs in comparison to crossovers (CO) could be detected in Arabidopsis. This indicates that NCOs happen either more rarely in plants or that the conversion tract length is significantly shorter than in other organisms. As the TOP3α/RMI1-mediated dissolution of recombination intermediates results exclusively in NCOs, we suggest that the peculiar GC pattern found in plants is connected to the unique role, members of the RTR complex play in plant meiosis.
    Frontiers in Plant Science 02/2014; 5:33. DOI:10.3389/fpls.2014.00033 · 3.95 Impact Factor
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