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Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and the...

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Autores principales: Shinohara, Takeshi, Ikawa, Shukuko, Iwasaki, Wakana, Hiraki, Toshiki, Hikima, Takaaki, Mikawa, Tsutomu, Arai, Naoto, Kamiya, Nobuo, Shibata, Takehiko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2015
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333409/
https://www.ncbi.nlm.nih.gov/pubmed/25561575
http://dx.doi.org/10.1093/nar/gku1364
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author Shinohara, Takeshi
Ikawa, Shukuko
Iwasaki, Wakana
Hiraki, Toshiki
Hikima, Takaaki
Mikawa, Tsutomu
Arai, Naoto
Kamiya, Nobuo
Shibata, Takehiko
author_facet Shinohara, Takeshi
Ikawa, Shukuko
Iwasaki, Wakana
Hiraki, Toshiki
Hikima, Takaaki
Mikawa, Tsutomu
Arai, Naoto
Kamiya, Nobuo
Shibata, Takehiko
author_sort Shinohara, Takeshi
collection PubMed
description In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.
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spelling pubmed-43334092015-03-18 Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair Shinohara, Takeshi Ikawa, Shukuko Iwasaki, Wakana Hiraki, Toshiki Hikima, Takaaki Mikawa, Tsutomu Arai, Naoto Kamiya, Nobuo Shibata, Takehiko Nucleic Acids Res Genome Integrity, Repair and Replication In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. Oxford University Press 2015-01-30 2015-01-05 /pmc/articles/PMC4333409/ /pubmed/25561575 http://dx.doi.org/10.1093/nar/gku1364 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Genome Integrity, Repair and Replication
Shinohara, Takeshi
Ikawa, Shukuko
Iwasaki, Wakana
Hiraki, Toshiki
Hikima, Takaaki
Mikawa, Tsutomu
Arai, Naoto
Kamiya, Nobuo
Shibata, Takehiko
Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair
title Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair
title_full Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair
title_fullStr Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair
title_full_unstemmed Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair
title_short Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair
title_sort loop l1 governs the dna-binding specificity and order for reca-catalyzed reactions in homologous recombination and dna repair
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333409/
https://www.ncbi.nlm.nih.gov/pubmed/25561575
http://dx.doi.org/10.1093/nar/gku1364
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