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ABSTRACT The broadly conserved ParB protein performs crucial functions in bacterial chromosome segregation and replication regulation. The cellular function of ParB requires it to dimerize, recognizeparSDNA sequences, clamp on DNA, then slide to adjacent sequences through nonspecific DNA binding. How ParB coordinates nonspecific DNA binding and sliding remains elusive. Here, we combine multiplein vitrobiophysical and computational tools andin vivoapproaches to address this question. We found that the five conserved lysine residues in the C-terminal domain of ParB play distinct roles in proper positioning and sliding on DNA, and their integrity is crucial for ParB’sin vivofunctions. Many proteins with diverse cellular activities need to move on DNA while loosely bound. Our findings reveal the detailed molecular mechanism by which multiple flexible basic residues enable DNA binding proteins to efficiently slide along DNA.
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Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA
Abstract HU (Histone‐like protein fromEscherichia colistrain U93) is the most conserved nucleoid‐associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single‐molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface‐exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over‐condensed and mis‐segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid‐associated naRNAs, each previously implicated in HU’s high‐affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure‐binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.
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- Award ID(s):
- 1817551
- PAR ID:
- 10450003
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Microbiology
- Volume:
- 115
- Issue:
- 1
- ISSN:
- 0950-382X
- Page Range / eLocation ID:
- p. 12-27
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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