In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription. 

Dynamic ParB–DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation

In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.

/pdf/dynamic-parb-dna-interactions-initiate-and-maintain-a-9e1ylnmk.pdf

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome 'caretaker' that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these SSB proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Abstract The tripartite ParABS system mediates chromosome segregation in a wide range of bacteria. Dimeric ParB was proposed to nucleate on parS sites and spread to neighboring DNA. However, how properly distributed ParB dimers further compact chromosomal DNA into a higher-order nucleoprotein complex for partitioning remains poorly understood. Here, using a single-molecule approach, we show that tens of Bacillus subtilis ParB (Spo0J) proteins can stochastically multimerize on and stably bind to nonspecific DNA. The introduction of CTP promotes the formation and diffusion of the multimeric ParB along DNA, offering an opportunity for ParB proteins to further forgather and cluster. Intriguingly, ParB multimers can recognize parS motifs and are more inclined to remain immobile on them. Importantly, the ParB multimer features distinct capabilities of not only bridging two independent DNA molecules but also mediating their transportation, both of which are enhanced by the presence of either CTP or parS in the DNA. These findings shed new light on ParB dynamics in self-multimerization and DNA organization and help to better comprehend the assembly of the ParB-DNA partition complex.

/pdf/stochastically-multimerized-parb-orchestrates-dna-assembly-28zwbxy8.pdf

Stochastically multimerized ParB orchestrates DNA assembly as unveiled by single-molecule analysis

EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double‐stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding‐degradation coordination of an exonuclease. Here, using a single‐molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA–DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2‐mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4‐bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate‐limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.

Discrete RNA–DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate‐limiting steps

Significance Bloom syndrome helicase (BLM) is a multifunctional helicase that primarily catalyzes the separation of two single strands of DNA. Here, using a single-molecule optical tweezers approach combined with confocal microscopy, we monitored both the enzymatic activity and oligomeric status of BLM at the same time. Strikingly, a head-on collision of BLM-medicated DNA unwinding forks was found to effectively switch their oligomeric state and activity. Specifically, BLMs, upon collision, immediately fuse across the fork junctions and covert their activities from dsDNA unwinding to ssDNA translocation and protein displacement. These findings explain how BLM plays multiple functional roles in homologous recombination (HR). The single-molecule approach used here provides a reference model for investigating the relationship between protein oligomeric state and function.

The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition

DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.

/pdf/rnase-h1-facilitates-recombinase-recruitment-by-degrading-164zmnoq.pdf

RNase H1 facilitates recombinase recruitment by degrading DNA-RNA hybrids during meiosis.

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

Teng Wang

Author Tools

Chat about Author