Subcellular membrane-less organelles consist of proteins with low complexity domains. Many of them, such as hnRNPA1, can assemble into both a polydisperse liquid phase and an ordered solid phase of ...amyloid fibril. The former mirrors biological granule assembly, while the latter is usually associated with neurodegenerative disease. Here, we observe a reversible amyloid formation of hnRNPA1 that synchronizes with liquid-liquid phase separation, regulates the fluidity and mobility of the liquid-like droplets, and facilitates the recruitment of hnRNPA1 into stress granules. We identify the reversible amyloid-forming cores of hnRNPA1 (named hnRACs). The atomic structures of hnRACs reveal a distinct feature of stacking Asp residues, which contributes to fibril reversibility and explains the irreversible pathological fibril formation caused by the Asp mutations identified in familial ALS. Our work characterizes the structural diversity and heterogeneity of reversible amyloid fibrils and illuminates the biological function of reversible amyloid formation in protein phase separation.
BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. ...However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.
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.
SignificanceBloom 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.
Helicases are multifunctional motor proteins with the primary task of separating nucleic acid duplexes. These enzymes often exist in distinct oligomeric forms and play essential roles during nucleic ...acid metabolism. Whether there is a correlation between their oligomeric state and cellular function, and how helicases effectively perform functional switching remains enigmatic. Here, we address these questions using a combined single-molecule approach and Bloom syndrome helicase (BLM). By examining the head-on collision of two BLM-mediated DNA unwinding forks, we find that two groups of BLM, upon fork convergence, promptly oligomerize across the fork junctions and tightly bridge two independent single-stranded (ss) DNA molecules that were newly generated by the unwinding BLMs. This protein oligomerization is mediated by the helicase and RNase D C-terminal (HRDC) domain of BLM and can sustain a disruptive force of up to 300 pN. Strikingly, onsite BLM oligomerization gives rise to an immediate transition of their helicase activities, from unwinding dsDNA to translocating along ssDNA at exceedingly fast rates, thus allowing for the efficient displacement of ssDNA-binding proteins, such as RPA and RAD51. These findings uncover an activity transition pathway for helicases and help to explain how BLM plays both pro- and anti-recombination roles in the maintenance of genome stability.
BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. ...However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.
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