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 language | English |
|---|---|
| Title of host publication | Mechanisms of DNA Recombination and Genome Rearrangements |
| Subtitle of host publication | Methods to Study Homologous Recombination |
| Editors | Maria Spies, Anna Malkova |
| Publisher | Academic Press |
| Pages | 543-568 |
| Number of pages | 26 |
| Volume | 600 |
| ISBN (Print) | 9780128144299 |
| DOIs | |
| Publication status | Published - 2018 |
Publication series
| Name | Methods in Enzymology |
|---|---|
| Publisher | Academic Press |
| Volume | 600 |
| ISSN (Print) | 0076-6879 |
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Keywords
- Bioinformatics
- DNA repair
- Enzyme kinetics
- Four-way DNA junction
- GEN1
- Genetic recombination
- Nucleases
- X-ray crystallography
Cite this
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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 proceeding › Chapter (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 -