Uncovering nick DNA binding by LIG1 at the single-molecule level.

DNA ligases repair the strand breaks are made continually and naturally throughout the genome, if left unrepaired and allowed to persist, they can lead to genome instability in the forms of lethal double-strand (ds) breaks, deletions, and duplications. DNA ligase 1 (LIG1) joins Okazaki fragments during the replication machinery and seals nicks at the end of most DNA repair pathways. Yet, how LIG1 recognizes its target substrate is entirely missing. Here, we uncover the dynamics of nick DNA binding by LIG1 at the single-molecule level. Our findings reveal that LIG1 binds to dsDNA both specifically and non-specifically and exhibits diffusive behavior to form a stable complex at the nick. Furthermore, by comparing with the LIG1 C-terminal protein, we demonstrate that the N-terminal non-catalytic region promotes binding enriched at nick sites and facilitates an efficient nick search process by promoting 1D diffusion along the DNA. Our findings provide a novel single-molecule insight into the nick binding by LIG1, which is critical to repair broken phosphodiester bonds in the DNA backbone to maintain genome integrity.

Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA.

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here, we selected a target site on bacteriophage λ-DNA and used optical tweezers combined with fluorescence to provide mechanistic insight into wild type Cas12a and three engineered variants, where the specific dsDNA and the unspecific ssDNA cleavage are dissociated (M1 and M2) and a third one which nicks the target DNA (M3). At low forces wtCas12a and the variants display two main off-target binding sites, while on stretched dsDNA at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. The multiple binding events onto dsDNA at high tension do not lead to cleavage, which is observed on the target site at low forces when the DNA is flexible. In addition, activity assays also show that the preferential off-target sites for this crRNA are not cleaved by wtCas12a, indicating that λ-DNA is only severed at the target site. Our single molecule data indicate that the Cas12a scaffold presents singular mechanical properties, which could be used to generate new endonucleases with biomedical and biotechnological applications.

Dynamic actin cross-linking governs the cytoplasm's transition to fluid-like behavior.

Cells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments change in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly cross-linked, elastic networks to weakly cross-linked viscous networks. While the role of actin cross-linking in controlling actin network mechanics is well-characterized in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent intracellular viscoelastic properties of living cells using multifrequency excitation and in situ optical trap calibration. At long timescales in the intracellular environment, we observe that the cytoskeleton becomes fluid-like. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant cross-linker. A disease-causing point mutation (K255E) of the actin cross-linker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild-type (WT) and K255E+/- cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3× faster to the fluid-like regime while K255E+/- cells are not affected. These results indicate that dynamic actin cross-linking enables the cytoplasm to flow at long timescales.