More Like this

1. Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

   https://doi.org/10.1093/nar/gkaa1184  

   Xue, Chaoyou; Molnarova, Lucia; Steinfeld, Justin B; Zhao, Weixing; Ma, Chujian; Spirek, Mario; Kaniecki, Kyle; Kwon, Youngho; Beláň, Ondrej; Krejci, Katerina; et al (December 2020, Nucleic Acids Research)

   null (Ed.)

   Abstract RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity. 

   more » « less
2. Dynamic structure of T4 gene 32 protein filaments facilitates rapid noncooperative protein dissociation

   https://doi.org/10.1093/nar/gkad595  

   Cashen, Ben A; Morse, Michael; Rouzina, Ioulia; Karpel, Richard L; Williams, Mark C (July 2023, Nucleic Acids Research)

   Abstract Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32–ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32–ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication. 

   more » « less
3. Smc5/6 uses its head-NSE module to preferentially associate with ssDNA gaps and ss-dsDNA junctions

   Irvin, Elizabeth M; Park, Sophia T; Miller-Browne, Victoria; Lever, Rebecca; Li, Shibai; Waterman, Grace; Kaur, Parminder; Zhao, Xiaolan; Wang, Hong (July 2026, Nucleic Acids)

   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. 

   more » « less
4. The structure of human CST reveals a decameric assembly bound to telomeric DNA

   https://doi.org/10.1126/science.aaz9649  

   Lim, Ci Ji; Barbour, Alexandra T.; Zaug, Arthur J.; Goodrich, Karen J.; McKay, Allison E.; Wuttke, Deborah S.; Cech, Thomas R. (June 2020, Science)

   The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo–electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome’s organization of double-stranded DNA. 

   more » « less
5. The Escherichia coli clamp loader rapidly remodels SSB on DNA to load clamps

   https://doi.org/10.1093/nar/gkac1169  

   Newcomb, Elijah S; Douma, Lauren G; Morris, Leslie A; Bloom, Linda B (December 2022, Nucleic Acids Research)

   Abstract Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme–DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein–protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein–protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading. 

   more » « less