The 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. 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 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission.

Gene silencing by heterochromatin is proposed to occur in part from the ability of HP1 proteins to spread across large regions of the genome, compact the underlying chromatin and recruit repressive activities1–3. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation driven phase-separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets but other molecules such as the transcription factor TFIIB show no preference. Using single-molecule DNA curtains we find that unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, though with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands based on nuclear context.

Highlights d Chromatin undergoes liquid-liquid phase separation (LLPS) under physiologic conditions d Linker DNA length and patterning, histone H1, and acetylation modulate chromatin LLPS d Acetylated chromatin only phase separates upon binding multi-bromodomain proteins d LLPS could enable establishment and maintenance of distinct chromatin compartments

Summary Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair ssDNA with a homologous dsDNA template. Here we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real-time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a 9th nucleotide coincides with an additional reduction in binding free energy and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination.

Summary 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 singlestranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent upon the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes, and supports a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

DNA strand exchange plays a central role in genetic recombination across all kingdoms of life, but the physical basis for these reactions remains poorly defined. Using single-molecule imaging, we found that bacterial RecA and eukaryotic Rad51 and Dmc1 all stabilize strand exchange intermediates in precise three-nucleotide steps. Each step coincides with an energetic signature (0.3 kBT) that is conserved from bacteria to humans. Triplet recognition is strictly dependent on correct Watson-Crick pairing. Rad51, RecA, and Dmc1 can all step over mismatches, but only Dmc1 can stabilize mismatched triplets. This finding provides insight into why eukaryotes have evolved a meiosis-specific recombinase. We propose that canonical Watson-Crick base triplets serve as the fundamental unit of pairing interactions during DNA recombination.

The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutSα and MutLα as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutSα as it searches for DNA lesions, MutLα as it searches for lesion-bound MutSα, and the MutSα/ MutLα complex as it scans the flanking DNA. We also show that MutLα undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutSα, but this activity is suppressed upon association with MutSα, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion-and ATP-dependent transitions involving both MutSα and MutLα, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA. P ostreplicative mismatch repair (MMR) corrects errors in DNA synthesis before they lead to genomic instability (1-3). MMR increases the fidelity of DNA replication up to 1,000-fold, and MMR defects in humans cause hereditary nonpolyposis colorectal cancer and may influence the onset of other tumors (1). MutSα and MutLα are conserved eukaryotic protein complexes necessary for MMR. MutSα is responsible for recognition of mismatches and small insertion/deletion loops (1-3), whereas MutLα harbors an endonuclease activity necessary for cleavage of the lesion-bearing DNA strand (4, 5).The challenges faced during MMR can be illustrated by considering that Saccharomyces cerevisiae should incur only approximately two mismatches per cell cycle (6). MutSα must find these rare lesions, MutLα must search for lesion-bound MutSα, and the lesion-bound MutSα/MutLα complex must search the flanking DNA for signals that distinguish the parental and daughter strands (1-3). Models describing how DNA-binding proteins search for specific targets include 3D diffusion (i.e., jumping), 1D hopping, 1D sliding, and intersegmental transfer; the latter three are categorized as facilitated diffusion because they allow target association rates exceeding limits imposed by 3D diffusion (7-10). New single-molecule and NMR techniques have led to resurgent interest in understanding how proteins locate targets (11-13), and using single-molecule imaging we previously demonstrated that MutSα and MutLα can undergo facilitated diffusion on undamaged DNA through 1Dsliding and 1D hopping, respectively (14, 15). However, no single-molecule study has directly revealed proteins searching for and subsequently engaging a target site through 1D diffusion (i.e., 1D sliding or 1D hopping) (7), and the inability to visualize target capture also prevents investigation of questions regarding downstream MMR events.Here we used nano...