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Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V H 3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V H and V L domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

CSB/ERCC6 belongs to an orphan subfamily of SWI2/SNF2-related chromatin remodelers and plays crucial roles in gene expression, DNA damage repair, and the maintenance of genome integrity. The molecular basis of chromatin remodeling by Cockayne syndrome B protein (CSB) is not well understood. Here we investigate the molecular mechanism of chromatin remodeling by Rhp26, a Schizosaccharomyces pombe CSB ortholog. The molecular basis of chromatin remodeling and nucleosomal epitope recognition by Rhp26 is distinct from that of canonical chromatin remodelers, such as imitation switch protein (ISWI). We reveal that the remodeling activities are bidirectionally regulated by CSB-specific motifs: the N-terminal leucine-latch motif and the C-terminal coupling motif. Rhp26 remodeling activities depend mainly on H4 tails and to a lesser extent on H3 tails, but not on H2A and H2B tails. Rhp26 promotes the disruption of histone cores and the release of free DNA. Finally, we dissected the distinct contributions of two Rhp26 C-terminal regions to chromatin remodeling and DNA damage repair., Competing Interests: The authors declare no conflict of interest.

Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11-Rad50-Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre11 2 -Rad50 2 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid., Competing Interests: The authors declare no conflict of interest.

CSB/ERCC6 (Cockayne syndrome B protein/excision repair cross-complementation group 6), a member of a subfamily of SWI2/SNF2 (SWItch/sucrose nonfermentable)-related chromatin remodelers, plays crucial roles in gene expression and the maintenance of genome integrity. Here, we report the mechanism of the autoregulation of Rhp26, which is the homolog of CSB/ERCC6 in Schizosaccharomyces pombe. We identified a novel conserved protein motif, termed the "leucine latch," at the N terminus of Rhp26. The leucine latch motif mediates the autoinhibition of the ATPase and chromatin-remodeling activities of Rhp26 via its interaction with the core ATPase domain. Moreover, we found that the C terminus of the protein counteracts this autoinhibition and that both the N- and C-terminal regions of Rhp26 are needed for its proper function in DNA repair in vivo. The presence of the leucine latch motif in organisms ranging from yeast to humans suggests a conserved mechanism for the autoregulation of CSB/ERCC6 despite the otherwise highly divergent nature of the N- and C-terminal regions.