Biology subjects

Szakacs, G.

Publications and source records attributed to Szakacs, G..

3 recordsLinked to original sources

bioRxiv2023

DNA-dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes

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 hSSB2s 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.

biochemistry↗

bioRxiv2023

Redox-dependent condensation and cytoplasmic granulation by human ssDNA binding protein 1 delineate roles in oxidative stress response

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. hSSB1s 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.

biochemistry↗

bioRxiv2022

Nucleotide binding is the critical regulator of ABCG2 conformational transitions

ABCG2 is an exporter type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

biochemistry↗