Organization of Chromatin by Intrinsic and Regulated Phase Separation.

Eukaryotic chromatin is highly condensed but dynamically accessible to regulation and organized into subdomains. We demonstrate that reconstituted chromatin undergoes histone tail-driven liquid-liquid phase separation (LLPS) in physiologic salt and when microinjected into cell nuclei, producing dense and dynamic droplets. Linker histone H1 and internucleosome linker lengths shared across eukaryotes promote phase separation of chromatin, tune droplet properties, and coordinate to form condensates of consistent density in manners that parallel chromatin behavior in cells. Histone acetylation by p300 antagonizes chromatin phase separation, dissolving droplets in vitro and decreasing droplet formation in nuclei. In the presence of multi-bromodomain proteins, such as BRD4, highly acetylated chromatin forms a new phase-separated state with droplets of distinct physical properties, which can be immiscible with unmodified chromatin droplets, mimicking nuclear chromatin subdomains. Our data suggest a framework, based on intrinsic phase separation of the chromatin polymer, for understanding the organization and regulation of eukaryotic genomes.

Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

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.

DNA sequence alignment by microhomology sampling during homologous recombination.

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 single-strand DNA (ssDNA) with a homologous double-strand DNA (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 ninth 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.

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. 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 molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although 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 on the basis of nuclear context.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

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.

Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor.

In physiological settings, all nucleic acids motor proteins must travel along substrates that are crowded with other proteins. However, the physical basis for how motor proteins behave in these highly crowded environments remains unknown. Here, we use real-time single-molecule imaging to determine how the ATP-dependent translocase RecBCD travels along DNA occupied by tandem arrays of high-affinity DNA binding proteins. We show that RecBCD forces each protein into its nearest adjacent neighbor, causing rapid disruption of the protein-nucleic acid interaction. This mechanism is not the same way that RecBCD disrupts isolated nucleoprotein complexes on otherwise naked DNA. Instead, molecular crowding itself completely alters the mechanism by which RecBCD removes tightly bound protein obstacles from DNA.

Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair.

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.

How do proteins locate specific targets in DNA?

Many aspects of biology depend on the ability of DNA-binding proteins to locate specific binding sites within the genome. Interest in this target search problem has been reinvigorated through the recent development of microscopy-based technologies capable of tracking individual proteins in real-time as they search for binding sites. In this review we discuss how two different proteins, lac repressor and RNA polymerase, have solved the target search problem through seemingly different mechanisms, with an emphasis on how recent in vitro single-molecule studies have influenced our understanding of these reactions.

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility-defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine-DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

Dynamic asymmetry and why chromatin defies simple physical definitions.

Recent experiments have demonstrated a nucleus where chromatin is molded into stable, interwoven loops. Yet, many of the proteins, which shape chromatin structure, bind only transiently. In those brief encounters, these dynamic proteins temporarily crosslink chromatin loops. While, on the average, individual crosslinks do not persist, in the aggregate, they are sufficient to create and maintain stable chromatin domains. Owing to the asymmetry in size and speed of molecules involved, this type of organization imparts unique biophysical properties-the slow (chromatin) component can exhibit gel-like behaviors, whereas the fast (protein) component allows domains to respond with liquid-like characteristics.

HP1 proteins compact DNA into mechanically and positionally stable phase separated domains.

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1ß, 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 HP1ß. 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.

MBD5 and MBD6 couple DNA methylation to gene silencing through the J-domain protein SILENZIO.

DNA methylation is associated with transcriptional repression of eukaryotic genes and transposons, but the downstream mechanism of gene silencing is largely unknown. Here we describe two Arabidopsis methyl-CpG binding domain proteins, MBD5 and MBD6, that are recruited to chromatin by recognition of CG methylation, and redundantly repress a subset of genes and transposons without affecting DNA methylation levels. These methyl-readers recruit a J-domain protein, SILENZIO, that acts as a transcriptional repressor in loss-of-function and gain-of-function experiments. J-domain proteins often serve as co-chaperones with HSP70s. Indeed, we found that SILENZIO's conserved J-domain motif was required for its interaction with HSP70s and for its silencing function. These results uncover an unprecedented role of a molecular chaperone J-domain protein in gene silencing downstream of DNA methylation.

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

DNA RECOMBINATION. Base triplet stepping by the Rad51/RecA family of recombinases.

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 promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion.

Gene expression, DNA replication and genome maintenance are all initiated by proteins that must recognize specific targets from among a vast excess of nonspecific DNA. For example, to initiate transcription, Escherichia coli RNA polymerase (RNAP) must locate promoter sequences, which compose <2% of the bacterial genome. This search problem remains one of the least understood aspects of gene expression, largely owing to the transient nature of search intermediates. Here we visualize RNAP in real time as it searches for promoters, and we develop a theoretical framework for analyzing target searches at the submicroscopic scale on the basis of single-molecule target-association rates. We demonstrate that, contrary to long-held assumptions, the promoter search is dominated by three-dimensional diffusion at both the microscopic and submicroscopic scales in vitro, which has direct implications for understanding how promoters are located within physiological settings.