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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...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2011
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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. |
format | Online Article Text |
id | pubmed-3241657 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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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