A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1

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Abstract

GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.

Original languageEnglish
Pages (from-to)11089-11098
Number of pages10
JournalNucleic Acids Research
Volume46
Issue number20
Early online date24 Sep 2018
DOIs
Publication statusPublished - 16 Nov 2018

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Ions
DNA
Enzymes
Substrate Specificity
Catalytic Domain
Chaetomium
Metals

Cite this

@article{2034e3b48be74e0fb8704e553d98d119,
title = "A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1",
abstract = "GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.",
author = "Yijin Liu and Freeman, {Alasdair D. J.} and Anne-C{\'e}cile D{\'e}clais and Lilley, {David M. J.}",
note = "Cancer Research UK [A18604]. Funding for open access charge: Departmental Fund. Conflict of interest statement. None declared.",
year = "2018",
month = "11",
day = "16",
doi = "10.1093/nar/gky863",
language = "English",
volume = "46",
pages = "11089--11098",
journal = "Nucleic Acids Research",
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publisher = "Oxford University Press",
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TY - JOUR

T1 - A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1

AU - Liu, Yijin

AU - Freeman, Alasdair D. J.

AU - Déclais, Anne-Cécile

AU - Lilley, David M. J.

N1 - Cancer Research UK [A18604]. Funding for open access charge: Departmental Fund. Conflict of interest statement. None declared.

PY - 2018/11/16

Y1 - 2018/11/16

N2 - GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.

AB - GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.

U2 - 10.1093/nar/gky863

DO - 10.1093/nar/gky863

M3 - Article

C2 - 30247722

VL - 46

SP - 11089

EP - 11098

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 20

ER -