The Bloom's syndrome helicase interacts directly with the human DNA mismatch repair protein hMSH6

Blooms syndrome (BS) is a rare genetic disorder characterised by genome instability and cancer susceptibility. BLM, the BS gene product, belongs to the highly-conserved RecQ family of DNA helicases. Although the exact function of BLM in human cells remains to be defined, it seems likely that BLM eliminates some form of homologous recombination (HR) intermediate that arises during DNA replication. Similarly, the mismatch repair (MMR) system also plays a crucial role in the maintenance of genomic stability, by correcting DNA errors generated during DNA replication. Recent evidence implicates components of the MMR system also in HR repair. We now show that hMSH6, a component of the heterodimeric mismatch recognition complex hMSH2/hMSH6 (hMutS?), interacts with the BLM protein both in vivo and in vitro. In agreement with these findings, BLM and hMSH6 colocalise to discrete nuclear foci following exposure of the cells to ionising radiation. However, the purified recombinant MutS? complex does not affect the helicase activity of BLM in vitro. As BLM has previously been shown to interact with the hMLH1 component of the hMLH1/hPMS2 (hMutL?) heterodimeric MMR complex, our present findings further strengthen the link between BLM and processes involving correction of DNA mismatches, such as in the regulation of the fidelity of homologous recombination events.</p

Identification of novel MORIPs.

1<p>MORIPs were identified using either the full-length mu-opioid receptor (MOR) or second intracellular loop of the MOR (IL2) as baits in MYTH or traditional Y2H screens, respectively.</p>2<p>Residues indicate the amino acid residues of the protein fragments recovered in the Y2H screens.</p

MORIP expression in brain regions of morphine-treated mice.

<p>Mice were treated for 96 hours with either morphine-containing (n = 5) or placebo (n = 4) pellets. Animals were sacrificed, brain regions dissected, and Western blots of selected MORIPs probed with MORIP-specific antibodies. Each panel contains a representative blot for a MORIP in the specified brain region (n = 4 blots/MORIP/brain region). Total protein was quantified by Ponceau stain of the blot prior to antibody probing. Bar graphs represent the average pixel density (as determined by imageJ) of four blots for each brain region normalized to total protein and placebo treatment. Data was analyzed using a two-sided Student’s t-test. Error is expressed as standard error of the mean. * indicates a statistically significant difference (p<.05) between sham and morphine treatment.</p

Role of SIAH1 and SIAH2 in regulating MOR ubiquitination.

<p>(A) Mapping the interaction site of MOR-IL2 on SIAH1 using GST-pulldowns. As controls, S-tagged constructs were incubated with untreated or GST-coated beads. (B) HEK-MOR cells were transfected with wild-type or truncated SIAH (trSIAH1 or trSIAH2) constructs and treated for 6 hours with 30 µM MG132. Proteins were immunoprecipitated and blots probed with either mouse anti-myc or mouse anti-HA to test for SIAH expression (left panels) and interaction with MOR (right panels), respectively. (C) HEK-MOR cells were transfected with wild-type or truncated SIAH constructs and either left untreated or treated with 10 µM DADLE for 6 hours. Blots were cut into sections and probed with rabbit anti-FLAG, mouse anti-myc, mouse anti-HA, or chicken anti-GAPDH antibodies. Bar graphs represent the average pixel density from 4 experiments normalized to GAPDH and untreated controls and subjected to a two-sided paired Student’s t-test. None of the SIAH constructs caused significant changes (at p<.05) in steady-state levels of MOR protein expression or in DADLE-mediated decreases in MOR expression levels. (D–F) Ubiquitination of MOR. Equal amounts of MOR (normalized from lysate blot) were loaded into immunoprecipitation reactions with anti-FLAG antibody. In each panel, the upper blot shows the IP probed for ubiquitin. All other blots show expression of various constructs or GAPDH in transfected cells. All experiments were performed in triplicate. (D) Steady-state ubiquitination levels in transfected HEK-MOR cells (E) Ubiquitination levels in transfected cells treated with 30 µM MG132 (F) Agonist induced ubiquitination in transfected cells treated with 10 µM DADLE.</p

Deletion of C443 affects stability of the D2R.

<p>(A, B) FLAG-tagged WT-D2R or ΔC443-D2R cDNAs were transiently expressed in HEK-293T cells. Quantitation of receptor expression was normalized to GAPDH expression for protein and mRNA. (A) Proteins were separated by SDS-PAGE and analyzed by Western blotting (left). WT-D2R and ΔC443-D2R mRNA expression levels were determined by RT-qPCR and normalized to wild-type levels (bottom graph). The top bar graph represents the average pixel density (as determined by ImageJ) from three separate experiments. All data were analyzed using a two-sided unpaired Student’s <i>t</i> test (expressed as ± SEM, <i>n</i> = 3, **P<0.01). (B) Cells were treated with 50 μg/mL cycloheximide for the indicated times. Proteins were separated by SDS-PAGE and analyzed by Western blotting (top). The bar graph represents the average pixel density (as determined by ImageJ) from four separate experiments. Data were analyzed using a two-sided unpaired Student’s <i>t</i> test (expressed as ± SEM, <i>n</i> = 4, *P < 0.05, **P<0.01).</p

Confirmation of MOR-MORIP interactions.

1<p>MORIPs isolated either in traditional or MYTH screens were tested for interaction with each of the intracellular loops (IL1, 2, or 3) or carboxyl-terminus (C-tail) of the MOR in a directed Y2H assay.</p>2<p>GST PD: Full-length MORIPs were tested for interaction with the MOR-IL2 in a GST pulldown assay.</p>3<p>Co-IP: Full-length MORIPs were tested for association with the delta (DOR), kappa (KOR), or mu (MOR) opioid receptor by co-immunoprecipitation.</p>+<p>indicates a positive result, - indicates a negative result, while ND indicates not done.</p

C443 is the major site of palmitoylation of the D2R.

<p>(A) Schematic representation of D2L showing the location of cysteine residue mutations. (B) FLAG-tagged WT-D2R or D2R mutants were transiently expressed in HEK-293T cells and expression in cell lysates was analyzed by Western blotting. Untransfected cells served as controls. (C) Normalized amounts of 15-HDYA-labeled D2Rs were IP’d and fluorescently imaged at 725 PMT (top) and 760 PMT (bottom). (D) Total D2R from IPs was analyzed by Western blotting. (E) Levels of palmitoylated D2Rs were normalized to the amount of IP’d receptor. Brackets indicate bands corresponding to D2R that were used for quantitation. The bar graph represents the average pixel density (as determined by ImageJ) from five separate experiments. Data were analyzed using a two-sided unpaired Student’s <i>t</i> test (expressed as ± SEM, <i>n</i> = 5, ***P < 0.001).</p

The Bloom's syndrome helicase interacts directly with the human DNA mismatch repair protein hMSH6

Blooms syndrome (BS) is a rare genetic disorder characterised by genome instability and cancer susceptibility. BLM, the BS gene product, belongs to the highly-conserved RecQ family of DNA helicases. Although the exact function of BLM in human cells remains to be defined, it seems likely that BLM eliminates some form of homologous recombination (HR) intermediate that arises during DNA replication. Similarly, the mismatch repair (MMR) system also plays a crucial role in the maintenance of genomic stability, by correcting DNA errors generated during DNA replication. Recent evidence implicates components of the MMR system also in HR repair. We now show that hMSH6, a component of the heterodimeric mismatch recognition complex hMSH2/hMSH6 (hMutS?), interacts with the BLM protein both in vivo and in vitro. In agreement with these findings, BLM and hMSH6 colocalise to discrete nuclear foci following exposure of the cells to ionising radiation. However, the purified recombinant MutS? complex does not affect the helicase activity of BLM in vitro. As BLM has previously been shown to interact with the hMLH1 component of the hMLH1/hPMS2 (hMutL?) heterodimeric MMR complex, our present findings further strengthen the link between BLM and processes involving correction of DNA mismatches, such as in the regulation of the fidelity of homologous recombination events.</p

Confirmation of MORIP/MOR-IL2 interactions.

<p>GST pulldown assays were performed to interrogate the interaction between selected full-length MORIPs and the MOR-IL2 domain. In the top three panels, MORIP-GST fusion proteins were used to pull down the S-tagged MOR-IL2. In the bottom three panels, MOR-IL2-GST fusion proteins were used to pull down S-tagged MORIPs. Pulldown products were purified on glutathione beads, separated by SDS-PAGE, and probed on Western blots using HRP-conjugated anti-S-tag antibodies. S-tagged MORIPs or MOR-IL2 domains produced in bacteria are shown in lysate lanes (Ly), while uncoated glutathione sepharose beads (Beads) or GST-coated glutathione sepharose beads (GST) incubated with S-tagged proteins served as negative controls. PD indicates pull-down lanes.</p

Co-localization of D2L and D2S with GFP-tagged PATs.

<p>D2 receptor constructs expressing D2L were co-expressed with GFP (A), GFP-GODZ (B), GFP-zDHHC4 (C, D), or GFP-zDHHC8 (E), followed by membrane permeabilization, fixation and immunofluorescent staining for D2L (red, left) and GFP (green, center) followed by confocal microscopy (right, co-localization in merged red and green channels evident in yellow). Two representative images are shown for low (C) and high (D) expression of GFP-zDHHC4.</p