Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of genome. SSBs share a conserved ssDNA-binding domain, less intrinsically disordered linker (IDL), highly C-terminal peptide (CTP) motif that mediates wide array protein-protein interactions with DNA-metabolizing proteins. Here we show Escherichia coli SSB protein forms liquid-liquid phase-separated condensates in cellular-like conditions through multifaceted involving all structural...

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which required for homology search, strand exchange and protection replication forks. nucleoprotein (NF) development genomic stability, its failure associated with developmental abnormalities tumorigenesis. Here we describe the structure human NFs Walker box mutants using electron microscopy. Wild-type adopt 'open' conformation when compared to a 'closed' formed by mutants, reflecting...

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate processes is thought to be achieved in part through differential recognition processing specific noncanonical structures, although mechanistic basis discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central recombination enzyme, exhibits substrates based on their geometry structure. Through...

Increasing evidence points to the importance of liquid-liquid phase separation (LLPS)-driven protein condensation in both eukaryotic and bacterial cell physiology. The formation condensates may involve interactions between structured (globular) domains intrinsically disordered regions requires multivalency that is often brought about by oligomerization. Here we dissect such contributions assessing engineered variants (Escherichia coli) single-stranded DNA binding (SSB) whose has recently...

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing protecting single-stranded (ss) as well organizing multiprotein complexes during replication, recombination, repair. Two mammalian SSB paralogs (hSSB1 hSSB2 in humans) were recently identified shown to be involved various genome maintenance processes. Following our recent discovery the liquid-liquid phase separation (LLPS) propensity Escherichia coli (Ec) SSB, here...

Abstract Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms DNA damage generation genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes may aid or impair genome stability faithful inheritance, underscoring importance quality control. Human Bloom’s syndrome (BLM, RecQ family) helicase plays central roles in pathway selection control unexplored molecular mechanisms. Here we show BLM’s multi-domain...

The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the activities some partners. One example is maintenance RecQ helicase. Here, we determine mechanistic details RecQ–SSB interaction using single-molecule magnetic tweezers rapid kinetic experiments. Our results...

Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication. These tethers form spontaneously many cell types, including of the immune and nervous systems. Traffic viral proteins, vesicles, calcium ions, mRNA, miRNA, mitochondria, lysosomes membrane proteins/raft domains have all been reported so far via open ended tunneling (TNTs). Recently we on existence plasma derived GM1/GM3 ganglioside enriched microvesicles costimulatory...

Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is key player in homologous recombination (HR)-based error-free repair processes.During HR, BLM exerts various biochemical activities including single-stranded (ss) translocation, separation and annealing complementary strands, disruption complex structures (e.g.displacement loops) contributes to quality control HR via clearance Rad51 nucleoprotein filaments.We performed quantitative mechanistic analysis truncated constructs...

Abstract DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain human Bloom’s syndrome (BLM) helicase was shown interact with RecA core, raising possibility that it may affect coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or...

RecQ helicases promote genomic stability through their unique ability to suppress illegitimate recombination and resolve intermediates. These DNA structure-specific activities of are mediated by the helicase-and-RNAseD like C-terminal (HRDC) domain, via unknown mechanisms. Here, employing single-molecule magnetic tweezers rapid kinetic approaches we establish that HRDC domain stabilizes intrinsic, sequence-dependent, pauses core helicase (lacking HRDC) in a geometry-dependent manner. We...

Abstract RecQ helicases—also known as the “guardians of genome”—play crucial roles in genome integrity maintenance through their involvement various DNA metabolic pathways. Aside from being conserved bacteria to vertebrates, importance is also reflected fact that humans impaired function multiple helicase orthologs are cause severe sets problems, including Bloom, Werner, or Rothmund-Thomson syndromes. Our aim was create and characterize a zebrafish ( Danio rerio ) disease model for Bloom...

Abstract Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components machinery and plays a crucial role in genome maintenance processes. However, its detailed function still not fully understood. Here we show human interacts G4 structures provide evidence for contribution to processing. The absence results...

Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in repair. Here, we show that purified hSSB1 undergoes redox-dependent liquid-liquid phase separation (LLPS) the presence of or RNA, features are distinct from those LLPS by bacterial SSB. nucleoprotein droplets form under physiological ionic conditions response to treatment modeling cellular oxidative stress. hSSB1's intrinsically disordered region is indispensable for LLPS, whereas all three cysteine...

The integrity of the genetic material is crucial for every organism. One intrinsic attack to genome stability stalling replication fork which can result in DNA breakage. Several factors, such as lesions or formation stable secondary structures (eg, G-quadruplexes) lead stalling. G-quadruplexes (G4s) are well-characterized that form within specific single-stranded sequence motifs and have been shown block/pause machinery. In most genomes several helicases described regulate G4 unfolding...

ABSTRACT Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing protecting single-stranded (ss) as well organizing multiprotein complexes during replication, recombination, repair. Two mammalian SSB paralogs (hSSB1 hSSB2 in humans) were recently identified shown to be involved various genome maintenance processes. Following our recent discovery the liquid-liquid phase separation (LLPS) propensity E. coli (Ec) SSB, here...

DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen-based replication system for simultaneous examination these tolerance processes. Using both Sanger next-generation sequencing combined with lesion-specific qPCR efficiency studies, demonstrate that works well studying NER TLS, especially its...

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Abstract Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location frequency these complexes is important for cellular function, genomic stability, number clinically anticancer antibacterial drugs, e.g., quinolones. We developed simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type topo in vitro . Using SHAN-seq, we detected Escherichia coli...

Abstract Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location frequency these complexes is important for cellular function, genomic stability number clinically anticancer antibacterial drugs, e.g. quinolones. We developed simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type topo in vitro. Using SHAN-seq, we detected Escherichia coli gyrase...