No abstract

Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin’s mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within the C-helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.

Mutagens constantly threaten the integrity of genetic material by causing damage in the form of double‐strand breaks (DSBs). Such damage is typically repaired through various modes of homologous recombination (HR). This project focuses on a form of HR known as single‐strand annealing (SSA), which is triggered when a DSB is located between two DNA repeats. This pathway generates 3' overhanging single‐stranded DNA “flaps” which are cleaved by the Rad1‐Rad10 endonuclease, aided by the mediator protein Saw1 in S. cerevisiae. Recent literature has demonstrated that Saw1‐dependent recruitment of Rad1‐Rad10 to damage sites, and SSA repair proficiency, are both a function of flap length. In this study, we set out to elucidate the precise flap length threshold requiring Saw1 for optimal SSA repair in vivo through fluorescence microscopy and real‐time quantitative PCR (qPCR). In order to investigate the importance of DNA flap length on Rad1‐Rad10 and Saw1 recruitment, we designed yeast strains generating 20, 30, or 50 nt flaps during SSA. A DSB was induced at a fluorescently labeled site in the yeast genome (DSB‐RFP), allowing us to monitor the recruitment of Rad10‐YFP via fluorescence microscopy. The percentages of cells containing colocalized foci in strains either wild‐type or mutant in Saw1 were quantified, revealing that Saw1‐mediated recruitment of the Rad1‐Rad10 complex increased with flap length. Surprisingly, we found that Saw1 was actively recruited to the DSB site across all flap length substrates via experiments with a Saw1‐CFP strain. Analyses via qPCR were also carried out to quantify SSA repair products formed in WT and saw1Δstrains. The data show diminished repair product formation in the absence of Saw1 as flap lengths increased. Together, these results suggest that Saw1 is required for Rad1‐Rad10 recruitment and efficient SSA repair in longer flap substrates with ~20–30 nt representing the key size triggering Saw1‐dependence for repair. Current efforts are underway to determine the Saw1‐dependence of proficient SSA repair under conditions in which background induction is not amplified by qPCR. Support or Funding Information The authors thank our Department and the NIH SC3 program for funding.

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1–ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and β subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin’s mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within this helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.