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 language | English |
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Pages (from-to) | 11089-11098 |
Number of pages | 10 |
Journal | Nucleic Acids Research |
Volume | 46 |
Issue number | 20 |
Early online date | 24 Sep 2018 |
DOIs | |
Publication status | Published - 16 Nov 2018 |
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A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1. / Liu, Yijin; Freeman, Alasdair D. J.; Déclais, Anne-Cécile; Lilley, David M. J. (Lead / Corresponding author).
In: Nucleic Acids Research, Vol. 46, No. 20, 16.11.2018, p. 11089-11098.Research output: Contribution to journal › Article
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 -