Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

1 Citation (Scopus)

Abstract

Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.

Original languageEnglish
Title of host publicationMechanisms of DNA Recombination and Genome Rearrangements
Subtitle of host publicationMethods to Study Homologous Recombination
EditorsMaria Spies, Anna Malkova
PublisherAcademic Press
Pages543-568
Number of pages26
Volume600
ISBN (Print)9780128144299
DOIs
Publication statusPublished - 2018

Publication series

NameMethods in Enzymology
PublisherAcademic Press
Volume600
ISSN (Print)0076-6879

Fingerprint

Cruciform DNA
Structural properties
Enzymes
Chaetomium
DNA
Eukaryotic Cells
Bioinformatics
Crystallization
Computational Biology
Fungi
Crystal lattices
Genetic Recombination
Purification
Agglomeration

Keywords

  • Bioinformatics
  • DNA repair
  • Enzyme kinetics
  • Four-way DNA junction
  • GEN1
  • Genetic recombination
  • Nucleases
  • X-ray crystallography

Cite this

Liu, Y., Freeman, A., Déclais, A-C., Gartner, A., & Lilley, D. M. J. (2018). Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes. In M. Spies, & A. Malkova (Eds.), Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination (Vol. 600, pp. 543-568). (Methods in Enzymology; Vol. 600). Academic Press. https://doi.org/10.1016/bs.mie.2017.11.021
Liu, Yijin ; Freeman, Alasdair ; Déclais, Anne-Cécile ; Gartner, Anton ; Lilley, David M. J. / Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes. Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination. editor / Maria Spies ; Anna Malkova. Vol. 600 Academic Press, 2018. pp. 543-568 (Methods in Enzymology).
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abstract = "Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.",
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Liu, Y, Freeman, A, Déclais, A-C, Gartner, A & Lilley, DMJ 2018, Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes. in M Spies & A Malkova (eds), Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination. vol. 600, Methods in Enzymology, vol. 600, Academic Press, pp. 543-568. https://doi.org/10.1016/bs.mie.2017.11.021

Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes. / Liu, Yijin; Freeman, Alasdair; Déclais, Anne-Cécile; Gartner, Anton; Lilley, David M. J. (Lead / Corresponding author).

Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination. ed. / Maria Spies; Anna Malkova. Vol. 600 Academic Press, 2018. p. 543-568 (Methods in Enzymology; Vol. 600).

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

TY - CHAP

T1 - Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes

AU - Liu, Yijin

AU - Freeman, Alasdair

AU - Déclais, Anne-Cécile

AU - Gartner, Anton

AU - Lilley, David M. J.

N1 - No funding info

PY - 2018

Y1 - 2018

N2 - Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.

AB - Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized.

KW - Bioinformatics

KW - DNA repair

KW - Enzyme kinetics

KW - Four-way DNA junction

KW - GEN1

KW - Genetic recombination

KW - Nucleases

KW - X-ray crystallography

U2 - 10.1016/bs.mie.2017.11.021

DO - 10.1016/bs.mie.2017.11.021

M3 - Chapter (peer-reviewed)

C2 - 29458774

AN - SCOPUS:85041113881

SN - 9780128144299

VL - 600

T3 - Methods in Enzymology

SP - 543

EP - 568

BT - Mechanisms of DNA Recombination and Genome Rearrangements

A2 - Spies, Maria

A2 - Malkova, Anna

PB - Academic Press

ER -

Liu Y, Freeman A, Déclais A-C, Gartner A, Lilley DMJ. Biochemical and Structural Properties of Fungal Holliday Junction-Resolving Enzymes. In Spies M, Malkova A, editors, Mechanisms of DNA Recombination and Genome Rearrangements: Methods to Study Homologous Recombination. Vol. 600. Academic Press. 2018. p. 543-568. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2017.11.021