Direct association of Bloom's syndrome gene product with the human mismatch repair protein MLH1

Abstract

Bloom's syndrome (BS) is a rare genetic disorder characterised by genomic instability and cancer susceptibility. BLM, the gene mutated in BS, encodes a member of the RecQ family of DNA helicases. Here, we identify hMLH1, which is involved in mismatch repair (MMR) and recombination, as a protein that directly interacts with BLM both in vivo and in vitro, and that the two proteins co-localise to discrete nuclear foci. The interaction between BLM and hMLH1 appears to have been evolutionarily conserved, as Sgs1p, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Mlh1p. However, cell extracts derived from BS patients show no obvious defects in MMR compared to wild-type- and BLM-complemented BS cell extracts. We conclude that the hMLH1-BLM interaction is not essential for post-replicative MMR, but, more likely, is required for some aspect of genetic recombination.

Figure 1

YTH interactions. (A) Schematic representation of BLM. The two acidic domains (striped), the helicase domain (black), the HRDC domain (stippled) and the two putative nuclear localisation signals (arrow) are indicated. The portion of BLM used as bait in the YTH screen is shown below. (B) Interactions of BLM and hMLH1 in the YTH assay. The L40 yeast strain was co-transformed with plasmids encoding the indicated proteins, and three independent colonies were grown on Trp^–Leu^–His^– selective plates prior to assessment of β-galactosidase activity. hMLH1(198–756) is the prey found in the YTH screen. Bait dependency is shown with a non-cognate protein (S.cerevisiae Rer2p) fused to LexAdbd. (C) YTH interactions of Sgs1p and yMlh1p as well as interspecies interactions. β-Galactosidase filter assay demonstrating the interaction of yMlh1p with a deletion mutant of Sgs1p (amino acids 784–1447) as well as with full-length Sgs1p and BLM and of Sgs1p (amino acids 784–1447) with hMlh1p. Also shown are two negative controls, the empty bait (LexAdbd) vector co-transformed with yMlh1 and and the empty prey (Gal4ad) vector together with the Sgs1 deletion mutant.

Figure 2

BLM and hMLH exist as a complex in human cells. (A) Co-immunoprecipitation of BLM with hMLH1. BLM could be immunoprecipitated with an anti-hMLH1 antibody from 200 µg nuclear extract of TK6 (wt) cells (lane 4), but not from HCT116 (BLM⁺ hMLH1^–) nuclear extract (lane 3). Immunoprecipitated proteins were visualised by western blot analysis with antibodies against hMLH1 (upper) or BLM (lower). (B) Co-immunoprecipitation of hMLH1 with BLM. hMLH1 was immunoprecipitated from 200 µg nuclear extract of BJAB cells with an anti-BLM antibody, but not with IgG. The immunoprecipitated proteins were detected with antibodies against BLM (upper) or hMLH1 (lower).

Figure 3

BLM and hMLH interact directly. (A) Purified human MutLα complex (1.5 µg), BSA (1 µg) and BLM (1 µg) were subjected to SDS–PAGE and stained with Coomassie blue. (B) Far western analysis. The proteins were transferred to a nitrocellulose membrane, renatured and incubated with purified MutLα complex (0.5 µg/ml), total Sf9 extracts expressing either hMLH1 or hPMS2 or purified recombinant BLM (1 µg/ml). Western blotting using anti-hMLH1, anti-hPMS2 (Ab-1; Calbiochem) and anti-BLM antibodies was used to detect the presence of the latter proteins on the membrane. The faster running bands in lane 10 are degradation products of hPMS2.

Figure 4

Interaction domain mapping of BLM and hMLH1. (A and B) Yeast two-hybrid assays. The sequence boundaries of the deletion mutants tested in a β-galactosidase filter assay are shown with the corresponding amino acid positions indicated on the left. The black bars indicate positive and striped bars negative interactions. (C and D) In vitro binding assays. (C) Aliquots of 0, 0.625, 1.25 and 2.5 pmol recombinant MutLα and BSA were spotted onto a nitrocellulose membrane and probed with 20 (*) or 40 µl (**), respectively, of the reaction mixture containing the indicated in vitro transcribed and translated BLM proteins. The autoradiogram of the gel shows 2 µl of the radiolabelled proteins used in the assay. (D) The same approach as in (C), but with immobilised full-length BLM and BSA on the membrane and the indicated in vitro transcribed and translated hMLH1 deletion mutants as probes.

Figure 5

Co-localisation of BLM and hMLH1 in the nucleus of WI-38/VA-13 cells. Indirect immunofluorescence of BLM (green) and hMLH1 (red) is shown in WI-38/VA-13 cells. The yellow colour results from overlap of the red and green foci. Nuclear DNA was revealed by staining with Hoechst 33258.

Figure 6

In vitro MMR efficiency of BS cell lines. (A) Western blot showing the absence of BLM protein in the BS cell lines. Aliquots of 25 µg of the indicated nuclear extracts were probed with anti-BLM antibody (IHIC33) and anti-XPB antibody [TFIIH p89 (S-19); Santa Cruz Biotechnology] as control. (B) MMR efficiency of the BLM-negative human fibroblast GM08505 and of the human lymphoblasts GM09960 and GM03403. The MMR-proficient MRC5 SV40 fibroblasts, TK6 lymphoblasts, PSNF5 cells (GM08505 stably transfected with BLM cDNA) and the MMR-deficient hMLH1^–/– colon cancer cell line HCT116 were used as controls. The MMR efficiencies of the two BS lymphoblasts complemented with purified recombinant BLM protein are also shown. The repair efficiency is expressed as fmol phagemid DNA cleaved by BglII in 30 min.