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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 variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. (A) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. (B) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.
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Figure 3: Wild-type EXO1 and its E109K variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. (A) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. (B) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.

Mentions: Rather than examine further individual interactions between EXO1 and purified MMR proteins MLH1 and PCNA, which EXO1 has also been reported to bind (11,13,15,19), we decided to test the functionality of the EXO1 variants in an in vitro MMR assay, in which a phagemid heteroduplex substrate carrying a single T/G mismatch and a strand discrimination signal (a single nick generated by Nt.BstNBI) 361 nucleotides 5′ from the mispaired T was incubated with extracts of human cells (29). In this assay, the mismatch makes the phagemid refractory to cleavage with SalI, but correction of the mismatch to C/G through EXO1-mediated degradation of the nicked T-strand and repair synthesis restores the restriction site (29). SalI/DraI digestion of the repaired phagemid recovered from the extract thus gives rise to four fragments of 1324, 1160, 694 and 19 bp, whereas the uncorrected phagemid is cleaved only by DraI into fragments of 2484, 694 and 19 bp (Figure 3A).


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 variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. (A) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. (B) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066805&req=5

Figure 3: Wild-type EXO1 and its E109K variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. (A) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. (B) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.
Mentions: Rather than examine further individual interactions between EXO1 and purified MMR proteins MLH1 and PCNA, which EXO1 has also been reported to bind (11,13,15,19), we decided to test the functionality of the EXO1 variants in an in vitro MMR assay, in which a phagemid heteroduplex substrate carrying a single T/G mismatch and a strand discrimination signal (a single nick generated by Nt.BstNBI) 361 nucleotides 5′ from the mispaired T was incubated with extracts of human cells (29). In this assay, the mismatch makes the phagemid refractory to cleavage with SalI, but correction of the mismatch to C/G through EXO1-mediated degradation of the nicked T-strand and repair synthesis restores the restriction site (29). SalI/DraI digestion of the repaired phagemid recovered from the extract thus gives rise to four fragments of 1324, 1160, 694 and 19 bp, whereas the uncorrected phagemid is cleaved only by DraI into fragments of 2484, 694 and 19 bp (Figure 3A).

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