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Zinc-finger recombinase activities in vitro

Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse ‘Z-sites’, which each comprise a central core sequence flanked by zinc-finger dom...

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Autores principales: Prorocic, Marko M., Wenlong, Dong, Olorunniji, Femi J., Akopian, Aram, Schloetel, Jan-Gero, Hannigan, Adèle, McPherson, Arlene L., Stark, W. Marshall
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241657/
https://www.ncbi.nlm.nih.gov/pubmed/21849325
http://dx.doi.org/10.1093/nar/gkr652
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author Prorocic, Marko M.
Wenlong, Dong
Olorunniji, Femi J.
Akopian, Aram
Schloetel, Jan-Gero
Hannigan, Adèle
McPherson, Arlene L.
Stark, W. Marshall
author_facet Prorocic, Marko M.
Wenlong, Dong
Olorunniji, Femi J.
Akopian, Aram
Schloetel, Jan-Gero
Hannigan, Adèle
McPherson, Arlene L.
Stark, W. Marshall
author_sort Prorocic, Marko M.
collection PubMed
description Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse ‘Z-sites’, which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs.
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spelling pubmed-32416572011-12-19 Zinc-finger recombinase activities in vitro Prorocic, Marko M. Wenlong, Dong Olorunniji, Femi J. Akopian, Aram Schloetel, Jan-Gero Hannigan, Adèle McPherson, Arlene L. Stark, W. Marshall Nucleic Acids Res Nucleic Acid Enzymes Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse ‘Z-sites’, which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs. Oxford University Press 2011-11 2011-08-17 /pmc/articles/PMC3241657/ /pubmed/21849325 http://dx.doi.org/10.1093/nar/gkr652 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Nucleic Acid Enzymes
Prorocic, Marko M.
Wenlong, Dong
Olorunniji, Femi J.
Akopian, Aram
Schloetel, Jan-Gero
Hannigan, Adèle
McPherson, Arlene L.
Stark, W. Marshall
Zinc-finger recombinase activities in vitro
title Zinc-finger recombinase activities in vitro
title_full Zinc-finger recombinase activities in vitro
title_fullStr Zinc-finger recombinase activities in vitro
title_full_unstemmed Zinc-finger recombinase activities in vitro
title_short Zinc-finger recombinase activities in vitro
title_sort zinc-finger recombinase activities in vitro
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241657/
https://www.ncbi.nlm.nih.gov/pubmed/21849325
http://dx.doi.org/10.1093/nar/gkr652
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