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Single molecule tracking reveals the role of transitory dynamics of nucleoid-associated protein HU in organizing the bacterial chromosome

View ORCID ProfileKelsey Bettridge, Subhash Verma, View ORCID ProfileXiaoli Weng, View ORCID ProfileSankar Adhya, View ORCID ProfileJie Xiao
doi: https://doi.org/10.1101/2019.12.31.725226
Kelsey Bettridge
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
3Laboratory of Biochemistry and Genetics, National Institute for Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Subhash Verma
2Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Xiaoli Weng
2Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Sankar Adhya
2Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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  • For correspondence: adhyas@mail.nih.gov xiao@jhmi.edu
Jie Xiao
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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  • For correspondence: adhyas@mail.nih.gov xiao@jhmi.edu
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Abstract

HU is the most conserved nucleoid-associated protein in eubacteria and has been implicated as a key player in global chromosome organization. The mechanism of HU-mediated nucleoid organization, however, remains poorly understood. Using single molecule tracking coupled with genetic manipulations, we characterized the dynamics of HU in live Escherichia coli cells. We found that native HU dimers bind and unbind chromosomal DNAs weakly and transitorily across the entire nucleoid volume but remain nucleoid-localized, reminiscent of random diffusion in a liquid phase-separated, membrane-less “macro-compartment” distinct from the remaining cytosol. Mutating three key surface lysine residues of HU nearly entirely abolished the weak and transitory interactions of HU with DNA and led to severe cell growth and DNA segregation defects, suggesting the importance of HU’s interactions with chromosomal DNA mediated by the positively charged surface. A conserved proline residue important for recognizing bent and cruciform DNAs such as that in recombination intermediates, similarly abolished HU’s rapid and transitory DNA interaction dynamics but had little impact on its apparent binding stability with nonspecific chromosomal DNAs. Interestingly, the proline residue appeared to be important for HUαβ dimer formation as mutating this residue makes HUαβ behave similarly to HUα2 dimers. Finally, we find that while prior evidence has found HU capable of depositing nucleoid-associated noncoding RNAs onto cruciform DNA structures, deletion of these specific naRNAs or inhibition of global transcription had a relatively minor effect on HU dynamics irrespective altered nucleoid compaction. Our results suggest a model of chromosome organization mediated by weak, transient interactions of HU, a substantial deviation from nucleoid-like proteins such as histones. Such collective sum of the numerous weak, transitory binding events of HU with nonspecific chromosome DNAs could generates a “force” to maintain a dynamic, fluid nucleoid with enough flexibility to rapidly facilitate global topological processes such as replication or nucleoid segregation.

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Posted December 31, 2019.
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Single molecule tracking reveals the role of transitory dynamics of nucleoid-associated protein HU in organizing the bacterial chromosome
Kelsey Bettridge, Subhash Verma, Xiaoli Weng, Sankar Adhya, Jie Xiao
bioRxiv 2019.12.31.725226; doi: https://doi.org/10.1101/2019.12.31.725226
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Single molecule tracking reveals the role of transitory dynamics of nucleoid-associated protein HU in organizing the bacterial chromosome
Kelsey Bettridge, Subhash Verma, Xiaoli Weng, Sankar Adhya, Jie Xiao
bioRxiv 2019.12.31.725226; doi: https://doi.org/10.1101/2019.12.31.725226

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