Discrete RNA-DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate-limiting steps.

EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding-degradation coordination of an exonuclease. Here, using a single-molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA-DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2-mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4-bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate-limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.

The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition.

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.