Mihály Kovács

SUMMARY: Mammalian single-stranded DNA binding protein 1 (SSB1) has been established as an essential component of genome stability in both human cells and mice. Moreover, SSB1 was recently implicated in cytoplasmic stress response by its involvement in Ras GTPase-activating protein-binding protein 1 (G3BP1)-containing cytoplasmic stress granules (SGs) upon various forms of stress. Here, we generated and analyzed human cellular knockout and rodent ischemia-reperfusion (I/R) models to define SSB1’s roles in cytoplasmic stress response. Analysis of wild-type as well as SSB1 and G3BP1 knockout human retinal pigment epithelial (RPE-1) cells shows stress-specific incorporation of SSB1 into SGs and a negative regulator role for SSB1 in SG dynamics under sublethal stress conditions. We find that SSB1 knockout measurably increases cellular sensitivity to oxidative stress but does not alter cell proliferation following mild acute stress. Moreover, we detect SSB1 efflux from the nucleus upon stress that is dependent upon the presence of G3BP1 in a stress-specific manner. In addition, using mouse and rat models we observe significant upregulation and robust cytoplasmic granulation of SSB1 upon renal ischemia-reperfusion stress, establishing SSB1’s involvement in complex organismal stress response in vivo . Together, our data demonstrate active involvement of SSB1 in cytoplasmic response to cellular stress and acute kidney injury, with implications for targeting stress response functions in cancerous versus non-cancerous contexts. HIGHLIGHTS: SSB1 is incorporated into cytoplasmic stress granules and negatively regulates stress granule assembly under sublethal stress conditions SSB1 shows stress- and G3BP1-dependent nuclear efflux SSB1 is upregulated and undergoes apical granulation in renal epithelial cells during renal ischemia–reperfusion injury

Single-stranded DNA binding (SSB) proteins are essential components of genome metabolism in both bacteria and eukaryotes. Recently demonstrated condensation propensities have placed SSB functions in a new context regarding the organization of nucleic acid-modifying complexes. In this work, we provide functional dissection of the condensation and partner binding properties of Escherichia coli (Ec) SSB via engineered modifications of its intrinsically disordered linker (IDL) region. We identify specific alterations in two glycine-rich regions as well as aromatic and/or positively charged residues of the IDL by which a broad-range, selective modification of condensation propensity and condensate thermal and chemical stability can be achieved, while leaving the single-stranded DNA and partner protein binding functions of SSB unchanged. AlphaFold 3-predicted structures of tetrameric wild-type and engineered EcSSB constructs identify multiple possible binding sites for the conserved C-terminal tip on the tetramer core of the IDL, establishing a link between condensation propensity and restrictions in IDL conformational dynamics. Besides defining the contributions of IDL-driven interactions to driving protein condensation, these results pave the way for the definition of in vivo roles of EcSSB condensation via genetic engineering and delineate ways for further development of liquid–liquid phase separation prediction algorithms.

Increasing evidence points to the importance of liquid–liquid phase separation (LLPS)‐driven protein condensation in both eukaryotic and bacterial cell physiology. The formation of condensates may involve interactions between both structured (globular) domains and intrinsically disordered protein regions and requires multivalency that is often brought about by oligomerization. Here we dissect such contributions by assessing engineered variants of bacterial (Escherichia coli) single‐stranded DNA binding (SSB) protein whose condensation has recently been implicated in bacterial genome metabolism. A truncated SSB variant (SSBdC, lacking the conserved C‐terminal peptide (CTP)) was used to assess the importance of interactions between SSB's globular oligonucleotide/oligosaccharide binding (OB) domain and the CTP. We show that OB–CTP interactions are essential for dynamic condensation in physiological (crowded, glutamate‐rich) environments. Via assessment of a protein variant (SSB^H55Y) from the known thermosensitive ssb‐1 mutant, we also show that the perturbation of OB–OB contacts significantly impairs the stability of SSB tetramers and results in thermally induced protein aggregation, underscoring the importance of multivalence brought about by stereospecific contacts. Our data point to adaptive fine‐tuning of SSB interactions to physiological condensation and demonstrate that SSB represents a versatile system for selective engineering of condensation‐driving interactions between globular and disordered regions.

Single‐stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single‐stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid–liquid phase separation (LLPS) propensity of Escherichia coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2's C‐terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB‐driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single‐stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB‐mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single-stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid-liquid phase separation (LLPS) propensity of E. coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2’s C-terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB-driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single-stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB-mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in the repair of DNA breaks and oxidized DNA lesions. Here we show that hSSB1 undergoes liquid-liquid phase separation (LLPS) that is redox-dependent and requires the presence of single-stranded DNA or RNA, features that are distinct from those of LLPS by bacterial SSB. hSSB1 nucleoprotein droplets form under physiological ionic conditions, in response to treatment resulting in cellular oxidative stress. hSSB1’s intrinsically disordered region (IDR) is indispensable for LLPS, whereas all three cysteine residues of the oligonucleotide/oligosaccharide-binding (OB) fold are necessary to maintain redox-sensitive droplet formation. Proteins interacting with hSSB1 show selective enrichment inside hSSB1 droplets, suggesting tight content control and recruitment functions for the condensates. While these features appear instrumental for genome repair, we also detected hSSB1 condensates in the cytoplasm in response to oxidative stress in various cell lines. hSSB1 condensates colocalize with stress granules, implying unexplored extranuclear roles in cellular stress response. Our results suggest novel, condensation-linked roles for hSSB1, linking genome repair and cytoplasmic defense.

RecQ helicases—also known as the “guardians of the genome”—play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner, or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish (Danio rerio) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis, and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51, however, blm appears to affect its function independent of tp53. Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom’s syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM’s multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM’s interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms.

RecQ helicases - also known as the ‘guardians of the genome’ - play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish ( Danio rerio ) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51 , however, blm appears to effect its function independent of tp53 . Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein−protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid−liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage. Significance Cells must rapidly and efficiently react to DNA damage to avoid its harmful consequences. Here we report a molecular mechanism that gives rise to a model of how bacterial cells mobilize DNA repair proteins for timely response to genomic stress and initiation of DNA repair upon exposure of single-stranded DNA. We found that bacterial single-stranded DNA binding protein (SSB), a central player in genome metabolism, can undergo dynamic phase separation under physiological conditions. SSB condensates can store a wide array of DNA repair proteins that specifically interact with SSB. However, elevated levels of single-stranded DNA during genomic stress can dissolve SSB condensates, enabling rapid mobilization of SSB and SSB-interacting proteins to sites of DNA damage.