Sy Redding

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.

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.

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 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.

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.

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.

DNA dynamics and single-molecule biology.

DNA stores information. Its function as a universal genetic material is among the most highly conserved qualities of living things. Its system of four bases, when overlaid with spatial and temporal controls, governs biology across the entire scale of life, from enzymatic reactions inside E. coli to embryogenesis in humans. Because the primary function of DNA is to store and propagate information, its sequence is often taken to be its most important biochemical property. However, during the ordinary course of cellular activity, DNA must be manipulated in many ways as a physical structure—a polymer in solution—regardless of whether a specific sequence is relevant to a given process. The dominant functional unit of this manipulation in the cell is the interaction between DNA and protein. Some interactions access or even extract the underlying sequence content of the DNA, whereas many others are largely oblivious to that information. DNA’s primary function in storing genetic information requires a degree of stability. Indeed, the cell’s ability to correctly maintain and propagate DNA sequences through many cycles of duplication at low error rates is the source of both the continuity and the variability so fundamental to evolution.1 DNA is also constrained by the more immediate and practical concerns of the cell, which, in handling and passing on genetic content, must efficiently carry out duplication of chromosomes.2 The two intertwined factors of long-term stability and duplication efficiency are served by the fairly uniform global structure of DNA. The familiar double helix is generally not taken to vary significantly in structure along its length, and this is true on average—for example, across the span of a large genomic fragment. However, the physical properties of DNA are not uniform across regions of a size relevant to protein binding.3 Nor are they uniform across cellular environments, which are heterogeneous and fluctuating,2a,4 or even across experimental conditions.5 Appreciating the basic physical qualities of DNA as a function of physiologically and experimentally relevant variables is essential to understanding protein–DNA interactions, and the physical properties of DNA are particularly important when interpreting the results of single-molecule experiments. Important factors to consider are the polymer nature of DNA and the effect of various forces on this polymer, the effect of temperature and salt concentration, DNA length, and sequence-dependent variations in structure. Furthermore, DNA dynamics influence complex, subtle, and time-dependent protein behaviors on short spatial and temporal scales, a swath of which are only accessible through single-molecule methodologies. (Recent reviews that explore the nature of single-molecule experiments include refs (6a−6c)). In this review, we focus on illustrative examples from our laboratory that have employed “DNA curtains”: parallel arrays of individual DNA molecules used to visualize the behavior of DNA-interacting proteins. However, we consider these concepts broadly applicable to single-molecule studies. Single-molecule bioscience is still coming of age, and given recent technical and conceptual advances, it is poised to tackle progressively more complex and physiologically relevant problems. However, designing and interpreting single-molecule experiments to study protein–DNA interactions often does not map directly onto comparable biochemical studies, and yet characterizing a particular system comprehensively requires data from as many approaches as possible. Constructing such cohesive understanding from disparate viewpoints, all directed toward an understanding of the same biological system, requires that each field possess a general framework to interpret results which can be universally applied to interpretation of phenomena as witnessed by that field. A successful framework should grant access to fundamental concepts about the biology underlying a given system, and it is these fundamental concepts that allow for a lucid dialogue among multiple fields. Here we present a framework that describes the relationship between DNA and DNA-binding proteins by considering the general factors that affect their interaction. Others have previously triangulated the relationships between DNA structure, DNA sequence, and protein–DNA interactions in various combinations, and the concepts presented here are informed by these earlier ideas.3a−3e

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.

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

Significance Chromosomes are crowded places, and any nucleic acid motor proteins that act on DNA must function within these crowded environments. How crowded environments affect motor protein behaviors remains largely unexplored. Here, we use single-molecule fluorescence microscopy to visualize the ATP-dependent motor protein RecBCD as it travels along crowded DNA molecules bearing long tandem arrays of DNA binding proteins. Our findings show that RecBCD can push through highly crowded protein arrays while evicting the proteins from DNA. This behavior on crowded DNA is distinct from a previously described mechanism by which RecBCD disrupts single isolated nucleoprotein complexes. These findings may provide insights into how other types of motor proteins travel along crowded nucleic acids. 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.

E. Coli RNA Polymerase Searches for Promoters through 3D Diffusion

Gene expression, DNA replication, and genome maintenance all start with site-specific DNA binding proteins, which must recognize specific targets from among a vast excess of nonspecific DNA. For example, to initiate transcription, E. coli RNA polymerase (RNAP) must locate promoter sequences, which comprise <2% of the bacterial genome. This promoter search problem remains one of the least understood aspects of gene expression, largely due to the transient nature of intermediates involved in the search process. Here we use single-molecule microscopy to visualize RNAP in real time as it searches for promoters, and we develop a theoretical framework that allows us to analyze target searches at the submicroscopic scale based on single-molecule promoter association rates. Contrary to long-held assumptions, we demonstrate that the promoter search by E. coli RNAP is dominated entirely by 3D diffusion, which has direct implications for understanding how E. coli RNAP and other proteins locate their targets within physiological settings.