Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Abstract

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 E. 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 SSBinteracting 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. RESULTS SSB forms dynamic LLPS condensates in physiologically relevant conditions in vitroDue to the fact that SSB shares several features with hitherto described LLPS drivers, we sought to determine if LLPS could be a yet undiscovered capability of SSB facilitating its multifaceted roles in genome maintenance. We performed a bioinformatics analysis that revealed that E. coli SSB, in particular its IDL region, shows high propensity for LLPS, according to multiple dedicated sequencebased prediction methods (Bolognesi et al., 2016;Lancaster et al., 2014;Vernon et al., 2018) (Fig. 1B-D). This feature of SSB is universally conserved among bacteria, as the majority of analyzed SSBs (72.1 %) harbored by representative bacterial species from 15 large phylogenetic groups show similar LLPS propensities (Tables S1-2; Fig. 1E).In line with its predicted LLPS propensity, purified E. coli SSB (30 µM; SSB concentrations are expressed as those of tetramer molecules throughout the paper) forms an opaque, turbid solution at low NaCl concentration (50 mM) in vitro at room temperature and also at the native temperature of E. coli cells (37°C) ( Fig. 2A-B). The process is reversible as elevation of NaCl concentration to 200 mM leads to immediate loss of turbidity. The Clconcentration in E. coli cells depends on their environment (Schultz et al., 1962); however, while the exact value is not known, it can be assumed to be in a similar range (5-100 mM) as that measured for the cells of mammalian E. coli hosts, whereas the major metabolic anion in bacterial cells is glutamate (Glu; 100 mM) (Bennett et al., 2009). Strikingly, SSB forms a turbid solution even in 200 mM NaGlu ( Fig. 2A-B). (It must be noted that experiments using NaGlu always contained a fixed amount of 20 mM NaCl, for technical reasons.) Investigation of the protein solution by differential interference contrast (DIC) microscopy after diluting SSB into low-salt buffer revealed the formation of regular spherical drop...

The emergence of phase separation as an organizing principle in bacteria

“…For a more in-depth review of BR-body function and phylogenetic distribution, see Muthunayake et al (4). (21) In vitro (18) In vitro (18) In vitro (18) In vitro (22) In vitro (22) In vitro (22) In vitro (47) In vitro (23) In vivo In vitro spherical (23,24,26) In situ icosahedral (95)(96)(97)(98) In situ (25,29) In vitro (23) In vivo (91) In vitro (26) In vitro (23,24,26) ND…”

“…Single-stranded DNA binding protein (SSB) stabilizes single-stranded DNA (ssDNA) and recruits proteins essential for DNA replication, repair, and recombination (49). This NAP is a good candidate for condensate formation through LLPS because it forms phase-separated droplets, is promiscuous in its binding interactions, and contains an IDR (22). A combination of turbidity measurements and fluorescence imaging found that SSB forms liquid-like droplets in vitro.…”

Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

“…Given Pol IV is constitutively elevated in our imaging strain, the increase in static Pol IV molecules near lesion-stalled replisomes is primarily due to decreased outflux of Pol IV from the replication fork. During replication a steady state level of SSB is present on the lagging strand, forming a condensate of SSB-Ct (Harami et al, 2019), yet individual SSB molecules are rapidly turned over because the complete synthesis of an Okazaki fragment takes only ~2 seconds, limiting the average lifetime of lagging-strand SSB molecules to around 1 second (Ogawa and Okazaki, 2002;Wu et al, 1992). This timescale may be too short to allow for association of Pol IV with SSB.…”

The emergence of phase separation as an organizing principle in bacteria

“…However, these behaviors are almost impossible to observe in tiny bacteria using conventional microscopy. Thus, bacterial condensate formation has been demonstrated by in vitro reconstitution, by visualizing the building blocks with fluorescence or differential interference contrast microscopy, and by correlating these LLPS activities in vitro with the behaviors of foci in cells (17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

“…Additionally, the in vitro concentrations and environmental conditions (salt concentrations, pH, temperature, crowding agents) under which components phase separate have been measured (17,19,(21)(22)(23)(24)(25)27). The liquid-like behavior of condensates has been tested by fluorescence recovery after photobleaching (FRAP) and by observing droplet fusion events (17,21,24,26,28).…”