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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]; [...]

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51–ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3–6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.Analyses of the structure and biochemical properties of the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 reveal details of its role in the repair of DNA double-strand breaks. [ABSTRACT FROM AUTHOR]

Homologous recombination is a mechanism for DNA repair that enables the exchange of genetic information between DNA molecules. Structural analysis reveals how the protein RecA orchestrates this process. Structural data reveal how RecA protein orchestrates recombination., Author(s): Upasana Roy, Eric C. Greene Author Affiliations: Demystifying the D-loop during DNA recombination Our chromosomes are subjected to a constant barrage of DNA-damaging agents that can lead to the [...]

In physiological settings, nucleic-acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyse protein displacement from RNA and DNA (1-4). However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multicolour single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase, holoenzymes, stalled elongation complexes and transcribing RNA polymerases in either head-to-head or head-to-tail orientations, as well as [EcoR.sup.IE111Q], lac repressor and even nucleo-somes. RecBCD did not pause during collisions and often pushed proteins thousands of base pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein-protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next., RecBCD is a heterotrimeric translocase involved in initiating homologous recombination and processing stalled replication forks (5,6).RecB is a 3' → 5' SF1A helicase and contains a nuclease domain for DNA [...]