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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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Structure, expression and purification of wild-type EXO1 and its E109K and D173A variants. (A) Structure of human EXO1 bound to DNA [3QEB.pdb; (36)]. Catalytic metal ions are depicted as purple spheres, the catalytic aspartate 173 is indicated in red and glutamate 109 in blue. (B) Recombinant EXO1 wild-type (wt), E109K (EK) and D173A (DA) variant proteins used in this study. The figure is a scan of a 4–15% SDS-polyacrylamide gel stained with Coomassie blue. M, pre-stained molecular size marker. (C) EXO1 wild-type and its E109K and D173A variants are devoid of non-specific endonucleolytic activity, as revealed by their inability to convert supercoiled phagemid DNA into a nicked, open circular form upon 30 min incubation with 10 nM variants. As a control, the plasmid was digested with NtBstNBI, a nickase with a single recognition site in this vector, or with DNaseI. Single-stranded phagemid DNA was included as a control.
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Figure 1: Structure, expression and purification of wild-type EXO1 and its E109K and D173A variants. (A) Structure of human EXO1 bound to DNA [3QEB.pdb; (36)]. Catalytic metal ions are depicted as purple spheres, the catalytic aspartate 173 is indicated in red and glutamate 109 in blue. (B) Recombinant EXO1 wild-type (wt), E109K (EK) and D173A (DA) variant proteins used in this study. The figure is a scan of a 4–15% SDS-polyacrylamide gel stained with Coomassie blue. M, pre-stained molecular size marker. (C) EXO1 wild-type and its E109K and D173A variants are devoid of non-specific endonucleolytic activity, as revealed by their inability to convert supercoiled phagemid DNA into a nicked, open circular form upon 30 min incubation with 10 nM variants. As a control, the plasmid was digested with NtBstNBI, a nickase with a single recognition site in this vector, or with DNaseI. Single-stranded phagemid DNA was included as a control.

Mentions: Exonuclease 1 belongs to the RAD2 family of structure-specific endonucleases (33), the N-terminal and internal nuclease domains of which are highly conserved from bacteriophage to man (33,34). EXO1 possesses 5′→3′ exonuclease and flap endonuclease activities (35) that share the same active site, composed of two Mg2+ atoms coordinated by five aspartate residues. In human EXO1, these are D30, D152, D171, D173 and D225 (36). Glutamate 109 is not part of the active site; it resides in a flexible loop between α-helices α4 and α5 (Figure 1A) that form, together with the β-sheet β3, a mobile microdomain, which was postulated to mediate protein/protein interactions (36). As the E109K mutation replaces a negatively charged residue with a positively charged one, we wanted to test whether it perturbs intermolecular protein/protein interactions between EXO1 and its partners during MMR.


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

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

Structure, expression and purification of wild-type EXO1 and its E109K and D173A variants. (A) Structure of human EXO1 bound to DNA [3QEB.pdb; (36)]. Catalytic metal ions are depicted as purple spheres, the catalytic aspartate 173 is indicated in red and glutamate 109 in blue. (B) Recombinant EXO1 wild-type (wt), E109K (EK) and D173A (DA) variant proteins used in this study. The figure is a scan of a 4–15% SDS-polyacrylamide gel stained with Coomassie blue. M, pre-stained molecular size marker. (C) EXO1 wild-type and its E109K and D173A variants are devoid of non-specific endonucleolytic activity, as revealed by their inability to convert supercoiled phagemid DNA into a nicked, open circular form upon 30 min incubation with 10 nM variants. As a control, the plasmid was digested with NtBstNBI, a nickase with a single recognition site in this vector, or with DNaseI. Single-stranded phagemid DNA was included as a control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Structure, expression and purification of wild-type EXO1 and its E109K and D173A variants. (A) Structure of human EXO1 bound to DNA [3QEB.pdb; (36)]. Catalytic metal ions are depicted as purple spheres, the catalytic aspartate 173 is indicated in red and glutamate 109 in blue. (B) Recombinant EXO1 wild-type (wt), E109K (EK) and D173A (DA) variant proteins used in this study. The figure is a scan of a 4–15% SDS-polyacrylamide gel stained with Coomassie blue. M, pre-stained molecular size marker. (C) EXO1 wild-type and its E109K and D173A variants are devoid of non-specific endonucleolytic activity, as revealed by their inability to convert supercoiled phagemid DNA into a nicked, open circular form upon 30 min incubation with 10 nM variants. As a control, the plasmid was digested with NtBstNBI, a nickase with a single recognition site in this vector, or with DNaseI. Single-stranded phagemid DNA was included as a control.
Mentions: Exonuclease 1 belongs to the RAD2 family of structure-specific endonucleases (33), the N-terminal and internal nuclease domains of which are highly conserved from bacteriophage to man (33,34). EXO1 possesses 5′→3′ exonuclease and flap endonuclease activities (35) that share the same active site, composed of two Mg2+ atoms coordinated by five aspartate residues. In human EXO1, these are D30, D152, D171, D173 and D225 (36). Glutamate 109 is not part of the active site; it resides in a flexible loop between α-helices α4 and α5 (Figure 1A) that form, together with the β-sheet β3, a mobile microdomain, which was postulated to mediate protein/protein interactions (36). As the E109K mutation replaces a negatively charged residue with a positively charged one, we wanted to test whether it perturbs intermolecular protein/protein interactions between EXO1 and its partners during MMR.

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