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Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1.

Bregenhorn S, Jiricny J - Nucleic Acids Res. (2014)

Bottom Line: Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome.We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates.In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from it.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cancer Research of the University of Zurich and the ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

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Wild-type EXO1 and its E109K and D173A variants interact directly with MSH2 and CtIP. (A) Far-western blot showing a direct interaction between EXO1 and MSH2. The MSH2/MSH6 (MutSα) heterodimer was separated by SDS-PAGE (left panel) and the MSH6 and MSH2 subunits were transferred onto a membrane, which was subsequently incubated with the recombinant EXO1 variants (right panels). Hybridization with an anti-EXO1 antibody revealed specific interactions of the variants with the MSH2 subunit of MutSα. BSA was run on the SDS-PAGE gel and used as a negative control for interaction with EXO1. M, pre-stained molecular size marker. (B) Far-western blot showing a direct interaction between EXO1 and CtIP. EXO1 wild-type and mutants were separated by SDS-PAGE (Coomassie staining top panel), transferred onto a membrane and incubated with recombinant FLAG-tagged CtIP. The interaction was revealed upon hybridization of the membrane with anti-FLAG to detect CtIP (bottom panel). M, pre-stained molecular size marker.
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Figure 2: Wild-type EXO1 and its E109K and D173A variants interact directly with MSH2 and CtIP. (A) Far-western blot showing a direct interaction between EXO1 and MSH2. The MSH2/MSH6 (MutSα) heterodimer was separated by SDS-PAGE (left panel) and the MSH6 and MSH2 subunits were transferred onto a membrane, which was subsequently incubated with the recombinant EXO1 variants (right panels). Hybridization with an anti-EXO1 antibody revealed specific interactions of the variants with the MSH2 subunit of MutSα. BSA was run on the SDS-PAGE gel and used as a negative control for interaction with EXO1. M, pre-stained molecular size marker. (B) Far-western blot showing a direct interaction between EXO1 and CtIP. EXO1 wild-type and mutants were separated by SDS-PAGE (Coomassie staining top panel), transferred onto a membrane and incubated with recombinant FLAG-tagged CtIP. The interaction was revealed upon hybridization of the membrane with anti-FLAG to detect CtIP (bottom panel). M, pre-stained molecular size marker.

Mentions: We then asked whether the EXO1 variants interacted with the MSH2 subunit of the mismatch recognition factor MutSα as reported previously (6,9,10,22). We separated the MSH6 and MSH2 subunits of purified recombinant MutSα on a denaturing polyacrylamide gel, electrotransferred the polypeptides onto a nitrocellulose membrane, denatured them in 6 M guanidinium hydrochloride, allowed them to renature and then incubated them with the recombinant EXO1 variants. Following extensive washing, we visualized the bound EXO1 variants with an anti-EXO1 antibody. BSA was used as the negative control. As shown in Figure 2A, all three variants interacted efficiently with MSH2 on these far-western blots.


Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1.

Bregenhorn S, Jiricny J - Nucleic Acids Res. (2014)

Wild-type EXO1 and its E109K and D173A variants interact directly with MSH2 and CtIP. (A) Far-western blot showing a direct interaction between EXO1 and MSH2. The MSH2/MSH6 (MutSα) heterodimer was separated by SDS-PAGE (left panel) and the MSH6 and MSH2 subunits were transferred onto a membrane, which was subsequently incubated with the recombinant EXO1 variants (right panels). Hybridization with an anti-EXO1 antibody revealed specific interactions of the variants with the MSH2 subunit of MutSα. BSA was run on the SDS-PAGE gel and used as a negative control for interaction with EXO1. M, pre-stained molecular size marker. (B) Far-western blot showing a direct interaction between EXO1 and CtIP. EXO1 wild-type and mutants were separated by SDS-PAGE (Coomassie staining top panel), transferred onto a membrane and incubated with recombinant FLAG-tagged CtIP. The interaction was revealed upon hybridization of the membrane with anti-FLAG to detect CtIP (bottom panel). M, pre-stained molecular size marker.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Wild-type EXO1 and its E109K and D173A variants interact directly with MSH2 and CtIP. (A) Far-western blot showing a direct interaction between EXO1 and MSH2. The MSH2/MSH6 (MutSα) heterodimer was separated by SDS-PAGE (left panel) and the MSH6 and MSH2 subunits were transferred onto a membrane, which was subsequently incubated with the recombinant EXO1 variants (right panels). Hybridization with an anti-EXO1 antibody revealed specific interactions of the variants with the MSH2 subunit of MutSα. BSA was run on the SDS-PAGE gel and used as a negative control for interaction with EXO1. M, pre-stained molecular size marker. (B) Far-western blot showing a direct interaction between EXO1 and CtIP. EXO1 wild-type and mutants were separated by SDS-PAGE (Coomassie staining top panel), transferred onto a membrane and incubated with recombinant FLAG-tagged CtIP. The interaction was revealed upon hybridization of the membrane with anti-FLAG to detect CtIP (bottom panel). M, pre-stained molecular size marker.
Mentions: We then asked whether the EXO1 variants interacted with the MSH2 subunit of the mismatch recognition factor MutSα as reported previously (6,9,10,22). We separated the MSH6 and MSH2 subunits of purified recombinant MutSα on a denaturing polyacrylamide gel, electrotransferred the polypeptides onto a nitrocellulose membrane, denatured them in 6 M guanidinium hydrochloride, allowed them to renature and then incubated them with the recombinant EXO1 variants. Following extensive washing, we visualized the bound EXO1 variants with an anti-EXO1 antibody. BSA was used as the negative control. As shown in Figure 2A, all three variants interacted efficiently with MSH2 on these far-western blots.

Bottom Line: Because not all LS families carry mutations in these four genes, the search for cancer-associated mutations was extended to genes encoding other members of the mismatch repairosome.We now report that, contrary to earlier reports, and unlike the catalytic site mutant D173A, the EXO1 E109K variant resembled the wild-type (wt) enzyme on all tested substrates.In the light of our findings, we attempt here to reinterpret the results of the phenotypic characterization of a knock-in mouse carrying the E109K mutation and cells derived from it.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cancer Research of the University of Zurich and the ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

Show MeSH
Related in: MedlinePlus