Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Single-molecule experiments reveal the elbow as an essential folding guide in SMC coiled-coil arms.

Structural maintenance of chromosome (SMC) complexes form ring-like structures through exceptional elongated coiled-coils (CCs). Recent studies found that variable CC conformations, including open and collapsed forms, which might result from discontinuities in the CC, facilitate the diverse functions of SMCs in DNA organization. However, a detailed description of the SMC CC architecture is still missing. Here, we study the structural composition and mechanical properties of SMC proteins with optical tweezers unfolding experiments using the isolated Psm3 CC as a model system. We find a comparatively unstable protein with three unzipping intermediates, which we could directly assign to CC features by crosslinking experiments and state-of-the-art prediction software. Particularly, the CC elbow is shown to be a flexible, potentially non-structured feature, which divides the CC into sections, induces a pairing shift from one CC strand to the other and could facilitate large-scale conformational changes, most likely via thermal fluctuations of the flanking CC sections. A replacement of the elbow amino acids hinders folding of the consecutive CC region and frequently leads to non-native misalignments, revealing the elbow as a guide for proper folding. Additional in vivo manipulation of the elbow flexibility resulted in impaired cohesin complexes, which directly link the sensitive CC architecture to the biological function of cohesin.

Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes.

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.