Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA

<i>E. coli</i> histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU–DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of...

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Autores principales:	Li Huang, Zhenfeng Zhang, Roger McMacken
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	HU DNA supercoiling affinity chemical crosslinking Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1cec0f66d91642efa482bce21fbe964f
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spelling	oai:doaj.org-article:1cec0f66d91642efa482bce21fbe964f2021-11-25T16:54:26ZInteraction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA10.3390/biom111117242218-273Xhttps://doaj.org/article/1cec0f66d91642efa482bce21fbe964f2021-11-01T00:00:00Zhttps://www.mdpi.com/2218-273X/11/11/1724https://doaj.org/toc/2218-273X<i>E. coli</i> histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU–DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity (<i>σ</i> = −0.06–0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA (<i>σ</i> = −0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5–6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.Li HuangZhenfeng ZhangRoger McMackenMDPI AGarticleHUDNA supercoilingaffinitychemical crosslinkingMicrobiologyQR1-502ENBiomolecules, Vol 11, Iss 1724, p 1724 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	HU DNA supercoiling affinity chemical crosslinking Microbiology QR1-502
spellingShingle	HU DNA supercoiling affinity chemical crosslinking Microbiology QR1-502 Li Huang Zhenfeng Zhang Roger McMacken Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA
description	<i>E. coli</i> histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU–DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity (<i>σ</i> = −0.06–0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA (<i>σ</i> = −0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5–6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.
format	article
author	Li Huang Zhenfeng Zhang Roger McMacken
author_facet	Li Huang Zhenfeng Zhang Roger McMacken
author_sort	Li Huang
title	Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA
title_short	Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA
title_full	Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA
title_fullStr	Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA
title_full_unstemmed	Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA
title_sort	interaction of the <i>escherichia coli</i> hu protein with various topological forms of dna
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/1cec0f66d91642efa482bce21fbe964f
work_keys_str_mv	AT lihuang interactionoftheiescherichiacoliihuproteinwithvarioustopologicalformsofdna AT zhenfengzhang interactionoftheiescherichiacoliihuproteinwithvarioustopologicalformsofdna AT rogermcmacken interactionoftheiescherichiacoliihuproteinwithvarioustopologicalformsofdna
_version_	1718412822982950912

Interaction of the <i>Escherichia coli</i> HU Protein with Various Topological Forms of DNA

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