BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome - PubMed (original) (raw)

BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome

Thiyam Ramsing Singh et al. Genes Dev. 2008.

Abstract

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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Figures

Figure 1.

Figure 1.

RMI2 is a novel component of the BTB complex. (A) Silver-stained gel showing the polypeptide bands purified from the nuclear extract of HT1080 cells transduced with vector alone (Mock) and cells expressing F-BLM-H, as described in Materials and Methods. The polypeptides identified by MS analysis are indicated by an arrow, and the asterisk (*) marks polypeptides also found in the mock purification. (B) Immunoblot of complexes purified from HeLa or HT1080 cells using anti-Flag M2 agarose. HeLa cells were transduced with vector alone or with vector that contained HF-RMI2. HT1080 cells stably expressed F-BLM-H. Immunoblots were probed with the antibodies indicated on the right. (C) Immunoblot of endogenous RMI2 complex immuno-isolated using anti-RMI2 antibodies raised against either MBP-RMI2 (RMI2-A) or GST-RMI2 (RMI2-B) fusions and probed with antibodies against known BTB members. RMI2 was detected with RMI2-A antibody on these immunoblots. (D) Silver-stained gel showing polypeptide bands purified from the nuclear extract of HeLa S3 cells transduced with vector alone (Mock) and cells stably expressing HF-RMI2. The RMI2 complex was purified using two different NaCl concentrations (250 and 400 mM, as indicated). Note that the unique polypeptide with molecular mass ∼27 kDa (denoted as HF-RMI2-L) was identified as a longer version of RMI2 that resulted from a leaky start site in the retroviral construct used. (E) Immunoblot of RMI2 complex purified using anti-Flag M2 agarose from HeLa cells stably expressing either the vector alone or HF-RMI2. Immunoblots were probed with antibodies against BTB members. The flow-through (FT) fraction is shown as a measure of depletion of BTB proteins along with HF-RMI2. Note that there is a substantial amount of BLM not depleted along with HF-RMI2, Topo IIIα, and RMI1.

Figure 2.

Figure 2.

RMI2 is essential for the integrity of the BTB complex. (A) Immunoblot showing levels of BTB members, RMI2, and actin in lysates from HeLa cells that were transfected with the indicated siRNA oligos for 72 h. HeLa cells transfected with a scrambled siRNA oligo were used a control. Asterisk (*) denotes a nonspecific crossreactive band detected by anti-RMI1 antibodies. (B) Immunoblot showing levels of BTB members and RMI2 in lysates from GM08505 (BLM−/−) cells (lane 1) and the same cells corrected with BLM cDNA and transfected with either the control siRNA (lane 2) or RMI2 siRNA (lane 3). (C) Immunoblot showing input (lanes 1–3) and anti-Flag M2 agarose immunoprecipitated complex (lanes 4–6) from HeLa S3 cells transduced with vector alone or GM0067 (wild-type) and GM08505 (BLM−/−) fibroblast cells transduced with HF-RMI2. (D) Immunoblot showing the emergence of a slower mobility species of BLM and RMI2 (lanes 2–3) after HEK293 cells stably expressing HF-RMI2 were treated with either taxol or nocodazole for 16 h, as compared with untreated cells. (E) Immunoblot showing the effect of λ-protein phosphatase treatment on the slower migrating form of RMI2. (F) Graph showing MMS survival curve of RMI2, BLM. and RMI1 knockdown cells. HEK293 cells were transfected with either scramble or siRNA oligos targeting either BLM, RMI1, or RMI2, and were subsequently treated with the indicated concentration of MMS. Visible colonies from 200 cells were counted after 10 d. The data represent the percent survival, as compared with untreated cells. Each experiment was independently repeated three times and representative data are shown. Each experiment was performed in triplicate and mean values are shown with standard deviations.

Figure 3.

Figure 3.

RMI2 is essential for the chromatin targeting of BLM in response to replication fork blockage. (A) Immunoblot showing that RMI2 and BLM form a tighter complex in response to DNA damage or replication blockage. HeLa cells stably expressing HF-RMI2 were left untreated (UN), or were treated with HU (1.5 mM) or MMC (100 ng/mL) for 16 h. Cells were lysed and HF-RMI2 was immunodepleted with anti-Flag M2 agarose. Immunoblot showing the levels of BTB members and RMI2 in input and flow-through (FT) fractions and in immunoprecipitates. Cell lysate from HeLa cells transduced with vector alone was used as a control. (B,C). Immunoblot showing the levels of BTB members and RMI2 in the following cellular fractions: cyto-nucleoplasmic (S100), nucleoplasmic (S300), and chromatin (P300) (see the Material and Methods for details). (B) Comparison between untreated (lanes 1–3) and HU treated (lanes 4–6) cellular fractions. (C) Comparison of the effect of a control siRNA (scramble) (lanes 1–3), RMI2 siRNA (lanes 4–6), or RMI1 siRNA (lanes 7–9) on the distribution of BLM, Topo IIIα, RMI1, and RMI2 to various fractions. Cells were treated with HU prior to fractionation, and H2A serves as a marker for the chromatin fraction. Asterisk (*) denotes a nonspecific crossreactive band detected by anti-RMI1 antibodies.

Figure 4.

Figure 4.

RMI2 regulates the assembly of BLM nuclear foci. (A) HF-RMI2 was expressed in HeLa cells. Foci assembled by this fusion protein and by BLM following exposure to 2 mM HU for 24 h were detected by immunofluorescence microscopy using anti-Flag and anti-BLM antibodies, respectively. HF-RMI2 foci were detected in red, while BLM foci were detected in green. A merged image shows colocalizing foci. The position of nuclei is indicated by a counterstain with DAPI (blue). Examples are shown for an untreated population of cells (top panels) and for cells treated with 1 mM HU for 24 h (bottom panels). (B) Examples of BLM foci in HeLa cells transfected with a scrambled control siRNA or with a siRNA that targeted RMI2 are shown. Cells were treated with 2 mM HU for 24 h prior to fixation and preparation for immunofluorescence microscopy. Bar, 10 μm. (C,D) Quantification of the assembly of BLM foci in HeLa (C) and HEK293 (D) cells. Cells were transfected with a scrambled control siRNA or with a siRNA that targeted RMI2 and were fixed for immunofluorescence microscopy at 96 h after transfection. Cells were either left untreated or were exposed to 2 mM HU for 24 h. The mean percentage of cells with five or more BLM foci from three counts of 150 or more cells each is shown with the standard deviation. Statistical significance (P < 0.01) using a _Z_-test for two sample proportions is indicated by an asterisk (*).

Figure 5.

Figure 5.

RMI2 stimulates dHJ dissolution activity of the BTB complex. (A) Purified RMI2/RMI1 complex, 3 μg, was analyzed by Coomassie Blue-stained SDS-PAGE. (B) Purified RMI2/RMI1-(C) complex, 3 μg, was resolved by SDS-PAGE and stained with Coomassie Blue. (C) BLM (5 μg) or Topo IIIα (5 μg) was incubated with or without RMI2/RMI1 complex (5 μg), and protein complexes were captured on amylose resin, which was washed and treated with SDS to elute bound proteins. The supernatant (S) that contained unbound proteins, wash (W), and SDS eluate (E) were analyzed by SDS-PAGE. (D) Experiment performed as in C, but protein complexes were captured on anti-Flag agarose. (*) IgG light chain dissociated from anti-Flag M2 agarose. (E) Time course of dHJ dissolution by combinations of BLM–Topo IIIα, RMI1, and RMI2. (BTB) BLM, Topo IIIα, and RMI1 combined. (F) Time course of dHJ dissolution by combinations of BLM–Topo IIIα, RMI1, and RMI2 from five independent experiments presented in the graph. The average values ± SEM. (G) dHJ dissolution by the BTB complex with or without RMI2 as a function of KCl concentration. The histogram shows the average levels of dissolution ± SEM. from four independent experiments.

Figure 6.

Figure 6.

Point mutations in the conserved residues of RMI2 that bind inefficiently to the BTB complex are unstable. (A, lanes 2,4) Immunoblot showing that siRMI2-3′UTR specifically depletes endogenous RMI2 and not the exogenously expressed HF-RMI2. (Lanes 1,3) Scrambled oligos were used as a control. (B) Immunoblot showing that RMI2 mutants K24A, W59A, and W135A were unstable, as compared with wild-type (WT) RMI2 or the K100A or K121A mutants. GFP was used as an internal control to show that mRNA expression is comparable in all the cells stably expressing HF-RMI2 variants. Immunoprecipitation coupled with immunoblotting to assay for the binding of the various variants to the BTB complex in HeLa cells (C) and HEK293 cells (D). (E) Immunoblot showing the levels of BTB members in HeLa cells depleted of the endogenous RMI2. Note that when the endogenous RMI2 was knocked down using siRMI2-3′UTR, HF-RMI2 mutants (K24A, W59A, and W135A), which could not bind to BTB complex, were unable to protect Topo IIIα and RMI1 from degradation (lanes 4,6,10), while wild type or the K100A could rescue Topo IIIα and RMI1 from degradation (lanes 2,8). Asterisk (*) denotes nonspecific crossreactive bands detected by anti-Topo IIIα and anti-RMI1 antibodies.

Comment in

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