Single-Molecule Analysis of the Improved Variants of the G-Quadruplex Recognition Protein G4P.

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity toward G4s than the previously published BG4 antibody. To get insight into G4P- G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the protein's affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics.

The DNA strand exchange protein RAD51 facilitates the central step in homologous recombination, a process fundamentally important for accurate repair of damaged chromosomes, restart of collapsed replication forks, and telomere maintenance. The active form of RAD51 is a nucleoprotein filament that assembles on single-stranded DNA (ssDNA) at the sites of DNA damage. The c-Abl tyrosine kinase and its oncogenic counterpart BCR-ABL fusion kinase phosphorylate human RAD51 on tyrosine residues 54 and 315. We combined biochemical reconstitutions of the DNA strand exchange reactions with total internal reflection fluorescence microscopy to determine how the two phosphorylation events affect the biochemical activities of human RAD51 and properties of the RAD51 nucleoprotein filament. By mimicking RAD51 tyrosine phosphorylation with a nonnatural amino acid, p-carboxymethyl-l-phenylalanine (pCMF), we demonstrated that Y54 phosphorylation enhances the RAD51 recombinase activity by at least two different mechanisms, modifies the RAD51 nucleoprotein filament formation, and allows RAD51 to compete efficiently with ssDNA binding protein RPA. In contrast, Y315 phosphorylation has little effect on the RAD51 activities. Based on our work and previous cellular studies, we propose a mechanism underlying RAD51 activation by c-Abl/BCR-ABL kinases.

The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain.

T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PHn-PHc), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.