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Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.
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This content will become publicly available on July 30, 2027
Smc5/6 uses its head-NSE module to preferentially associate with ssDNA gaps and ss-dsDNA junctions
Structural Maintenance of Chromosomes (SMC) complexes play critical roles in genome maintenance. While canonical SMCs, such as cohesin and condensin, organize the genome into 3D structures, Smc5/6 directly promotes genome replication and repair. Insights into Smc5/6 function have emerged from recent fluorescence-based single-molecule studies showing its binding to structures resembling replication and repair intermediates. To provide a higher-resolution view of this facet of Smc5/6′s function, we employed atomic force microscopy (AFM) to visualize the budding yeast Smc5/6 at nanometer-scale resolution and investigate how it interacts with short ssDNA gaps that frequently form during genome maintenance. We report that the free or DNA-bound Smc5/6 adopts a ‘bead-on-stick’ shape, with its head-NSE module separated from its hinge domain by a non-bending arm region. Smc5/6 predominantly uses its head-NSE module to engage with dsDNA, and mutating DNA-binding residues within this structural module shifts the complex toward a hinge-mediated DNA-binding mode. Importantly, Smc5/6 shows an intrinsic association preference for ssDNA gap regions and ss-dsDNA junction sites over the flanking dsDNA regions, predominantly through the head-NSE module. AFM visualization of Smc5/6 and its structural elements in preferential gap and junction DNA association provides a framework for our continued understanding of Smc5/6′s roles in genome maintenance.
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- Award ID(s):
- 2414612
- PAR ID:
- 10693054
- Publisher / Repository:
- Nucleic Acids Research
- Date Published:
- Journal Name:
- Nucleic Acids
- ISSN:
- 0550-3280
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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