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1. RPA engages telomeric G-quadruplexes more effectively than CST

   https://doi.org/10.1093/nar/gkad315  

   Olson, Conner L. ; Barbour, Alexandra T. ; Wieser, Thomas A. ; Wuttke, Deborah S. ( May 2023 , Nucleic Acids Research)

   G-quadruplexes (G4s) are a set of stable secondary structures that form within guanine-rich regions of single-stranded nucleic acids that pose challenges for DNA maintenance. The G-rich DNA sequence at telomeres has a propensity to form G4s of various topologies. The human protein complexes Replication Protein A (RPA) and CTC1-STN1-TEN1 (CST) are implicated in managing G4s at telomeres, leading to DNA unfolding and allowing telomere replication to proceed. Here, we use fluorescence anisotropy equilibrium binding measurements to determine the ability of these proteins to bind various telomeric G4s. We find that the ability of CST to specifically bind G-rich ssDNA is substantially inhibited by the presence of G4s. In contrast, RPA tightly binds telomeric G4s, showing negligible changes in affinity for G4 structure compared to linear ssDNAs. Using a mutagenesis strategy, we found that RPA DNA-binding domains work together for G4 binding, and simultaneous disruption of these domains reduces the affinity of RPA for G4 ssDNA. The relative inability of CST to disrupt G4s, combined with the greater cellular abundance of RPA, suggests that RPA could act as a primary protein complex responsible for resolving G4s at telomeres.

    

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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)

   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.

    

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3. The Escherichia coli clamp loader rapidly remodels SSB on DNA to load clamps

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

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

   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.

    

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4. CST does not evict elongating telomerase but prevents initiation by ssDNA binding

   https://doi.org/10.1093/nar/gkab942  

   Zaug, Arthur J. ; Lim, Ci Ji ; Olson, Conner L. ; Carilli, Maria T. ; Goodrich, Karen J. ; Wuttke, Deborah S. ; Cech, Thomas R. ( October 2021 , Nucleic Acids Research)

   The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

    

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5. 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. 

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