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. 2008 Sep;36(16):5166-79.
doi: 10.1093/nar/gkn498. Epub 2008 Aug 5.

The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase delta

Nives Selak  1 , Csanád Z Bachrati, Igor Shevelev, Tobias Dietschy, Barbara van Loon, Anette Jacob, Ulrich Hübscher, Joerg D Hoheisel, Ian D Hickson, Igor Stagljar
Affiliations

The Bloom's syndrome helicase (BLM) interacts physically and functionally with p12, the smallest subunit of human DNA polymerase delta

Nives Selak et al. Nucleic Acids Res. 2008 Sep.

Abstract

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL delta (hPOL delta). The hPOL delta enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL delta strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL delta. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL delta.

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Figures

Figure 1.
Figure 1.
BLM and p12 of hPOL δ interact in vitro and in vivo. (A) Left panel, far-western analysis. hPOL λ, total protein extract from Sf9 insect cells, and hPOL δ enzyme were subjected to SDS–PAGE, transferred to a nitrocellulose membrane, and were incubated with purified recombinant BLM. Anti-BLM antibodies were used to permit the detection of p12 as a novel BLM-interacting protein (lane 6). Molecular weight markers are also indicated on the left. Right panel, purified p12 subunit of hPOL δ was subjected to SDS–PAGE, transferred to nitrocellulose membrane, incubated with BLM, and subsequently probed with the anti-BLM antibody (lane 9). (B) Reciprocal far-western analysis. BSA, WRN and BLM (left panel) were hybridized with the purified p12 and probed with the anti-p12 antibody (right panel). Anti-p12 antibodies were used to confirm that p12 specifically binds to BLM (lane 6). (C) BLM and p12 of hPOL δ interact in the YTH assay. The L40 yeast reporter strain was co-transformed with plasmids encoding the indicated full-length ‘bait’ (LexA-DBD) and ‘prey’ (Gal4-AD) fusions. Two independent colonies were grown on SD agar plates lacking tryptophan and leucine, but containing X-gal, prior to assessment of β-galactosidase activity. Also shown are two negative controls, p12 co-transformed with an empty prey vector and BLM co-transformed with lamin C protein. The previously described BLM/hp150 (CAF-1) interaction (23) was used as a positive control. (D) BLM and hPOL δ form a complex in human cells. 293T cells were transiently transfected with FLAG-p12, and were synchronized in S phase using 1 mM HU. Nuclear extracts derived from either unsynchronized (lane 3) or S-phase synchronized cells (lane 4) were immunoprecipitated with the anti-FLAG antibody or control IgG, and were analysed by SDS–PAGE. One-tenth (50 µg) of the same nuclear extract was used as input control (lane 1). Immunoprecipitated FLAG-p12 and BLM were detected by western blotting using the anti-FLAG and anti-BLM antibody, respectively (lane 4). p125, the largest subunit of hPOL δ, was also efficiently co-immunoprecipitated using the same anti-FLAG antibody (lanes 3 and 4). Reciprocal co-IP is shown in the middle panel: lane 5, input; lane 6, IP with the control IgG; lane 7, IP with an anti-BLM antibody (C-18) from nuclear extracts derived from unsynchronized 293T cells; lane 8, IP with an anti-BLM antibody (C-18) from nuclear extracts derived from the S-phase synchronized 293T cells. The known BLM interacting protein, hp150 (CAF-1) was also efficiently co-immunoprecipitated using the same anti-BLM antibody (lanes 7 and 8). As a loading control for lanes 1–8, 50 µg of the corresponding nuclear extract was probed with an anti-PARP1 antibody. Right panel shows co-IP with anti-BLM antibody (C-18) from BS cell nuclear extracts (BS) and BS cells containing the BLM cDNA (BS + pBLM). p12 could be co-immunoprecipitated in the presence of BLM from the S-phase synchronized nuclear extracts (lane 12) but not in the absence of BLM (lane 11). Lanes 9 and 10 are the inputs of the two different nuclear extracts.
Figure 2.
Figure 2.
Interaction region mapping of BLM and p12. (A) Mapping of the BLM interaction region. The L40 yeast strain was co-transformed with plasmids encoding the indicated BLM fragments fused to Gal4-AD and the full-length p12 fused to LexA-DBD. Two independent colonies were grown on SD agar plates lacking tryptophan and leucine, but containing X-gal, prior to assessment of β-galactosidase activity. Blue coloration of colonies is a marker of interaction. Full length BLM is also shown, with a red bar indicating its conserved helicase domain, a blue bar indicating RQC and a black bar indicating the HRDC domain. Interactions between a given bait/prey pair were quantified by measurements of β-galactosidase activity. Values represent means ± SD of three independent experiments. (B) Mapping of the p12 interaction region. The L40 yeast strain was co-transformed with plasmids encoding the indicated p12 fragments fused to LexA-DBD and full length BLM fused to Gal4-AD. In both (A) and (B) the sequence boundaries of deletion mutants tested are shown with the corresponding amino acid positions indicated on the right. Values obtained from liquid β-galactosidase assay are shown on the right and represent means ± SD of three independent experiments.
Figure 3.
Figure 3.
The hPOL δ enzyme specifically stimulates the BLM-mediated unwinding of the replication fork substrate in a concentration-dependent manner. (A) A total of 1.3 nM BLM was pre-incubated with the exonuclease-defective hPOL δ enzyme in various concentrations (33.5, 16.8, 8.4, 4.2, 2.1, 1 and 0.5 nM; lanes 6–12, respectively) on ice for 3 min, and the samples were then warmed to 37°C. The unwinding reaction was initiated immediately by the addition of substrate and ATP. Flame symbol depicts heat-denatured substrate (lane 2), or BLM incubated with heat-denatured hPOL δ enzyme at the highest concentration of the titration range (33.5 nM; lane 5), as described earlier. (B) Quantification of data presented in (A). Data were normalized to the non-treated (lane 1, taken as 0%) and boiled (lane 2, taken as 100%) samples. Maximal stimulation (at 6 times above BLM basal helicase activity) was achieved with a 13 × molar excess of hPOL δ.
Figure 4.
Figure 4.
The small subunit of the hPOL δ enzyme, p12, is sufficient to stimulate BLM helicase activity. (A) A total of 1.3 nM BLM was pre-incubated with various concentrations of p12 (1609, 804.5, 402.25, 201.13, 100.56, 50.28, 25.14, 12.57, 6.29 and 3.14 nM; lanes 6–15, respectively) on ice for 3 min, and the samples were then warmed to 37°C. The unwinding reaction was initiated immediately by the addition of substrate and ATP. Controls and symbols are as in Figure 3. (B) Quantification of data from (A). (C) A total of 0.85 nM BLM was incubated at 37°C for 5 min alone (lanes 1–9), or with 20 nM hPOL δ (lanes 10–18), or 20 nM p12 (lanes 19–27). The unwinding reaction was then initiated by the addition of ATP and substrate. Samples were withdrawn at the time points indicated above the lanes. (D) Quantification of data from (C).
Figure 5.
Figure 5.
The stimulatory effect of p12 can be localized to a small peptide, spanning the region that binds BLM. (A) A total of 0.85 nM BLM was incubated at 37°C for 5 min alone (lanes 1–9), or with 20 nM p1271–100 (a peptide that shows no binding to BLM; lanes 10–18), full length p12 (lanes 19–27) or p1230–60 (a peptide that spans the binding region to BLM; lanes 28–36). Reactions were run as in Figure 4B. (B) Quantification of data from (A).
Figure 6.
Figure 6.
BLM stimulates hPOL δ strand displacement activity. (A) Ten nanogram of hPOL δ alone (lane 2) or in the presence of an increasing amount (10, 20 or 50 ng) of BLM (lanes 3–5), PCNA (lanes 7–9) or 50 ng of E. coli RecQ (lane 10) were tested in primer extension assays using the X-poly DNA template as described in Materials and methods section. Eighteen-nucleotide primer was 5′ end labeled. Lane 6 contains 50 ng of BLM alone and shows that BLM does not have DNA polymerase activity. Lane 1: substrate alone; positions of oligonucleotide size-markers are indicated. Schematic representation of the X-poly DNA template is shown. The limit of extension is indicated with the arrow. (B) Quantification of products longer than 34 nt from (A).
Figure 7.
Figure 7.
Dual staining for BLM and hPOL δ suggests co-localization in vivo, which is stimulated during replicative stress. (A) GM00637 cells were arrested with 2.5 mM HU, and were stained as described in Materials and methods section. A representative image showing punctate BLM (green, left) and hPOL δ (red, second from left) staining. Coincidence of the green and red signals (yellow signal) suggests co-localization of the two proteins. The right panel shows the nucleus of the same cell stained with the DNA dye Hoechst 33258. (B) GM00637 cells grown on coverslips without HU treatment and were pulse-labeled with 25 μM BrdU for 5 min, and then stained as described in Materials and methods section. Representative images show staining of the same nucleus for BLM in green (top left), for hPOL δ in red (top, second from left), for BrdU in blue (top, second from right). Phase contrast image of the same nucleus is depicted on the top right. In the bottom row, combined images of the individual stainings from the top row are shown. As expected, the BrdU signal correlates well with the signal for hPOLδ (magenta-color pattern second panel from bottom right). The coincidence of BLM signal with either BrdU (cyan signal in bottom left) or hPOLδ (yellow signal in second panel from bottom left) is less pronounced.
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