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GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes

Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way...

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Autores principales: Freeman, Alasdair D.J., Liu, Yijin, Déclais, Anne-Cécile, Gartner, Anton, Lilley, David M.J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270448/
https://www.ncbi.nlm.nih.gov/pubmed/25315822
http://dx.doi.org/10.1016/j.jmb.2014.10.008
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author Freeman, Alasdair D.J.
Liu, Yijin
Déclais, Anne-Cécile
Gartner, Anton
Lilley, David M.J.
author_facet Freeman, Alasdair D.J.
Liu, Yijin
Déclais, Anne-Cécile
Gartner, Anton
Lilley, David M.J.
author_sort Freeman, Alasdair D.J.
collection PubMed
description Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1 nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1.
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spelling pubmed-42704482014-12-22 GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes Freeman, Alasdair D.J. Liu, Yijin Déclais, Anne-Cécile Gartner, Anton Lilley, David M.J. J Mol Biol Article Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1 nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1. Elsevier 2014-12-12 /pmc/articles/PMC4270448/ /pubmed/25315822 http://dx.doi.org/10.1016/j.jmb.2014.10.008 Text en © 2014 The Authors. Published by Elsevier Ltd. https://creativecommons.org/licenses/by/3.0/This work is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/) .
spellingShingle Article
Freeman, Alasdair D.J.
Liu, Yijin
Déclais, Anne-Cécile
Gartner, Anton
Lilley, David M.J.
GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes
title GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes
title_full GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes
title_fullStr GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes
title_full_unstemmed GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes
title_short GEN1 from a Thermophilic Fungus Is Functionally Closely Similar to Non-Eukaryotic Junction-Resolving Enzymes
title_sort gen1 from a thermophilic fungus is functionally closely similar to non-eukaryotic junction-resolving enzymes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270448/
https://www.ncbi.nlm.nih.gov/pubmed/25315822
http://dx.doi.org/10.1016/j.jmb.2014.10.008
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