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Cohesin is essential for the hierarchical organization of the eukaryotic genome and plays key roles in many aspects of chromosome biology. The conformation of cohesin bound to DNA remains poorly defined, leaving crucial gaps in our understanding of how cohesin fulfills its biological functions. Here, we use single-molecule microscopy to directly observe the dynamic and functional characteristics of cohesin bound to DNA. We show that cohesin can undergo rapid one-dimensional (1D) diffusion along DNA, but individual nucleosomes, nucleosome arrays, and other protein obstacles significantly restrict its mobility. Furthermore, we demonstrate that DNA motor proteins can readily push cohesin along DNA, but they cannot pass through the interior of the cohesin ring. Together, our results reveal that DNA-bound cohesin has a central pore that is substantially smaller than anticipated. These findings have direct implications for understanding how cohesin and other SMC proteins interact with and distribute along chromatin.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

The tumour suppressor complex BRCA1BARD1 functions in the repair of DNA double-stranded breaks by homologous recombination. During this process, BRCA1BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumour suppressor complex, BRCA2PALB2, and the recombinase RAD51. Here, by examining purified wild-type and mutant BRCA1BARD1, we show that both BRCA1 and BARD1 bind DNA and interact with RAD51, and that BRCA1BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. We provide evidence that BRCA1 and BARD1 are indispensable for RAD51 stimulation. Notably, BRCA1BARD1 mutants with weakened RAD51 interactions show compromised DNA joint formation and impaired mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1BARD1 in homologous recombination, an attribute of the tumour suppressor complex that could be targeted in cancer therapy., Author(s): Weixing Zhao (corresponding author) [1]; Justin B. Steinfeld [2]; Fengshan Liang [1, 3, 4]; Xiaoyong Chen [3, 4]; David G. Maranon [5]; Chu Jian Ma [2]; Youngho Kwon [1]; [...]