hMRE11 deficiency leads to microsatellite instability and defective DNA mismatch repair

Abstract

DNA mismatch repair (MMR) is essential in the surveillance of accurate transmission of genetic information, and defects in this pathway lead to microsatellite instability and hereditary nonpolyposis colorectal cancer (HNPCC). Our previous study raised the possibility that hMRE11 might be involved in MMR through physical interaction with hMLH1. Here, we show that hMRE11 deficiency leads to significant increase in MSI for both mono- and dinucleotide sequences. Furthermore, RNA-interference-mediated hMRE11-knockdown in HeLa cells results in MMR deficiency. Analysis of seven HNPCC-associated hMLH1 missense mutations located within the hMRE11-interacting domain shows that four mutations (L574P, K618T, R659P and A681T) cause near-complete disruption of the interaction between hMRE11 and hMLH1, and two mutations (Q542L and L582V) cause a 30% reduction of protein interaction. These findings indicate that hMRE11 represents a functional component of the MMR pathway and the disruption of hMLH1-hMRE11 interaction could be an alternative molecular explanation for hMLH1 mutations in a subset of HNPCC tumours.

Figure 1

Generation of stable hMRE11-knockdown HeLa cells. Gene silencing of hMRE11 in HeLa cells was achieved by the use of small interfering RNAs. To generate HeLa cells stably displaying hMRE11-knockdown, two pmH1P-neo-based RNAi constructs encoding short hairpin RNAs were used to transfect HeLa cells. Neomycin-resistant clones were selected with 400 μg/ml G418, and 150 μg of cell extracts was analysed by immunoblotting. HeLa, parental control; 12 and 14, two representative stable cell lines.

Figure 2

Effects of hMRE11-knockdown on microsatellite instability (MSI). Two out-of-frame MSI reporters, pA-OF and pCA-OF, were used to transfect a roughly equivalent number of parental and hMRE11-knockdown HeLa cells (HeLa 14), as well as the hMLH1-deficient cell line H6 as a control. The number of cells that expressed GFP was determined by FACS analysis performed with a total of 20,000 cells for HeLa and HeLa 14, and a total of 10,000 cells for parental and transfected H6 cells.

Figure 3

Effects of hMRE11-knockdown on DNA mismatch repair (MMR). A 50 μg portion of nuclear extracts derived from parental HeLa (H) and HeLa 14 (Hⁱ) cells and 17.6 μg of a partially purified hMRE11-enriched HeLa nuclear fraction SS1 were used to perform MMR reactions with 5′ G-T and 3′ G-T mismatch substrates. Repaired products were distinguished from substrates by restriction digestion, and repair efficiency was scored by dividing repaired products with the total substrate DNA. (A) MMR reactions were performed as indicated. The hMRE11-enriched SS1 fraction is defective for both 3′ and 5′ repairs. (B) Average repair efficiencies and standard deviations (error bars) were determined from three independent data points. ^*P=0.003, ^**P=0.007, t-test.

Figure 4

Interaction domain mapping for hMLH1 and hMRE11. (A) The 756-amino-acid full-length hMLH1 and a series of deletion mutants were encoded in pVPd as VP16-AD fusion proteins. The average β-galactosidase (β-gal) activity units and standard errors (s.e.) from at least three independent data points are listed. L40 double transformant harbouring full-length hMRE11-BD plasmid and an empty pVPd vector showed a background β-gal activity reading of 2.60±0.12. (B) cDNA fragments encoding the 708-amino-acid full-length hMRE11 and a series of hMRE11 truncation mutants in the form of Gal4-AD fusions were used to examine protein interactions with the full-length hMLH1 in LexA-BD fusion form. The protein interaction was ascertained as described in (A). The L40 double transformant harbouring full-length hMLH1-BD plasmid and an empty pGADT7 vector showed a background β-gal activity reading of 4.59±0.42. The two naturally occurring hMRE11 mutations R633stop and N117S are indicated by ATLD1 and ATLD3, respectively. In (A,B), the black bars indicate positive protein interactions monitored by X-gal filter assay. (C,D) Characterization of interacting domains by far-western analysis. The T₇ tag was removed from the construct encoding hMRE11 aa 452–634 fragment. (C) Purified T₇ tagged hMLH1 aa 495–756 protein (3 μg) and BSA were immobilized directly on the same nitrocellulose membranes, which were then probed with either purified MRE11 aa 452–634 proteins or the corresponding crude preparation. The anti-hMRE11 and anti-T₇ antibodies were used to detect the presence of hMRE11 aa 452–634 and T₇-hMLH1 aa 495–756 proteins, respectively. (D) A reciprocal far-western analysis of purified, immobilized hMRE11 aa 452–634 protein and BSA. The membranes were incubated with T₇-hMLH1 aa 495–756 crude lysate or purified protein. The same antibodies as in (C) were used for detection.

Figure 5

Far-western analysis of the effects of hMLH1 HNPCC missense mutations on the hMLH1 and hMRE11 interaction. Membranes with purified, immobilized hMRE11 aa 452–634 protein and BSA were probed independently with crude lysate containing wild-type and various hMLH1 mutant proteins. Conventional western blot analysis was carried out using anti-T₇ antibody to detect the captured wild-type and mutant hMLH1 aa 495–756 proteins.