Mechanism of target site selection by type V-K CRISPR-associated transposases.

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

Mechanism of target site selection by type V-K CRISPR-associated transposases.

Unlike canonical CRISPR-Cas systems that rely on RNA-guided nucleases for target cleavage, CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to facilitate RNA-guided transposition of large genetic payloads. Type V-K CASTs offer several potential upsides for genome engineering, due to their compact size, easy programmability, and unidirectional integration. However, these systems are substantially less accurate than type I-F CASTs, and the molecular basis for this difference has remained elusive. Here we reveal that type V-K CASTs undergo two distinct mobilization pathways with remarkably different specificities: RNA-dependent and RNA-independent transposition. Whereas RNA-dependent transposition relies on Cas12k for accurate target selection, RNA-independent integration events are untargeted and primarily driven by the local availability of TnsC filaments. The cryo-EM structure of the untargeted complex reveals a TnsB-TnsC-TniQ transpososome that encompasses two turns of a TnsC filament and otherwise resembles major architectural aspects of the Cas12k-containing transpososome. Using single-molecule experiments and genome-wide meta-analyses, we found that AT-rich sites are preferred substrates for untargeted transposition and that the TnsB transposase also imparts local specificity, which collectively determine the precise insertion site. Knowledge of these motifs allowed us to direct untargeted transposition events to specific hotspot regions of a plasmid. Finally, by exploiting TnsB's preference for on-target integration and modulating the availability of TnsC, we suppressed RNA-independent transposition events and increased type V-K CAST specificity up to 98.1%, without compromising the efficiency of on-target integration. Collectively, our results reveal the importance of dissecting target site selection mechanisms and highlight new opportunities to leverage CAST systems for accurate, kilobase-scale genome engineering applications.

Repair of mismatched templates during Rad51-dependent Break-Induced Replication.

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3' end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt-the latter being the size of the in vivo templates-we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3' end. Mismatch incorporation depends on the 3'→ 5' proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3' end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ "chews back" the 3' end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3' end of the donor.

Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks.

DNA double strand breaks (DSBs) are among some of the most deleterious forms of DNA damage. Left unrepaired, they are detrimental to genome stability, leading to high risk of cancer. Two major mechanisms are responsible for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). The complex nature of both pathways, involving a myriad of protein factors functioning in a highly coordinated manner at distinct stages of repair, lend themselves to detailed mechanistic studies using the latest single-molecule techniques. In avoiding ensemble averaging effects inherent to traditional biochemical or genetic methods, single-molecule studies have painted an increasingly detailed picture for every step of the DSB repair processes.

Dynamic action of DNA repair proteins as revealed by single molecule techniques: Seeing is believing.

DNA repair is a highly dynamic process in which the actual damage recognition process occurs through an amazing dance between the DNA duplex containing the lesion and the DNA repair proteins. Single molecule investigations have revealed that DNA repair proteins solve the speed-stability paradox, of rapid search versus stable complex formation, by conformational changes induced in both the damaged DNA and the repair proteins. Using Rad4, XPA, PARP1, APE1, OGG1 and UV-DDB as examples, we have discovered how these repair proteins limit their travel on DNA, once a lesion is encountered through a process of anomalous diffusion. We have also observed how PARP1 and APE1, as well as UV-DDB and OGG1 or APE1, co-localize dynamically at sites near DNA damage. This review highlights how our group has greatly benefited from our productive collaborations with Sam Wilson's research group.

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

Human Condensin I and II Drive Extensive ATP-Dependent Compaction of Nucleosome-Bound DNA.

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.

Damage sensor role of UV-DDB during base excision repair.

UV-DDB, a key protein in human global nucleotide excision repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a noncanonical role in base excision repair (BER). We investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase ß-gap filling activity by 30-fold. Single-molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moves to sites of 8-oxoG repair in cells, and UV-DDB depletion sensitizes cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.

Studying protein-DNA interactions using atomic force microscopy.

Atomic force microscopy (AFM) has made significant contributions to the study of protein-DNA interactions by making it possible to topographically image biological samples. A single protein-DNA binding reaction imaged by AFM can reveal protein binding specificity and affinity, protein-induced DNA bending, and protein binding stoichiometry. Changes in DNA structure, complex conformation, and cooperativity, can also be analyzed. In this review we highlight some important examples in the literature and discuss the advantages and limitations of these measurements. We also discuss important advances in technology that will facilitate the progress of AFM in the future.

PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1.

PARP1-dependent poly-ADP-ribosylation (PARylation) participates in the repair of many forms of DNA damage. Here, we used atomic force microscopy (AFM) and single molecule fluorescence microscopy to examine the interactions of PARP1 with common DNA repair intermediates. AFM volume analysis indicates that PARP1 binds to DNA at nicks, abasic (AP) sites, and ends as a monomer. Single molecule DNA tightrope assays were used to follow the real-time dynamic behavior of PARP1 in the absence and presence of AP endonuclease (APE1) on AP DNA damage arrays. These experiments revealed that PARP1 conducted damage search mostly through 3D diffusion. Co-localization of APE1 with PARP1 on DNA was found capable of inducing 1D diffusion of otherwise nonmotile PARP1, while excess APE1 also facilitated the dissociation of DNA-bound PARP1. Moreover, auto-PARylation of PARP1 allowed the protein to switch its damage search strategy by causing a 3-fold increase in linear diffusion. Finally, we demonstrated that PARP inhibitor olaparib did not significantly alter the rate of PARP1 dissociation from DNA, but instead resulted in more motility of DNA-bound PARP1 molecules.

Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair.

Optical Control of DNA Helicase Function through Genetic Code Expansion.

Nucleotide excision repair (NER) is a general DNA repair mechanism that is capable of removing a wide variety of DNA lesions induced by physical or chemical insults. UvrD, a member of the helicase SF1 superfamily, plays an essential role in bacterial NER by unwinding the duplex DNA in the 3' to 5' direction to displace the lesion-containing strand. In order to achieve conditional control over NER, we generated a light-activated DNA helicase. This was achieved through a site-specific incorporation of a genetically encoded hydroxycoumarin lysine at a crucial position in the ATP-binding pocket of UvrD. The resulting caged enzyme was completely inactive in several functional assays. Moreover, enzymatic activity of the optically triggered UvrD was comparable to that of the wild-type protein, thus demonstrating excellent OFF to ON switching of the helicase. The developed approach provides optical control of NER, thereby laying a foundation for the regulation of ATP-dependent helicase functions in higher organisms. In addition, this methodology is applicable to the light-activation of a wide range of ATPases.

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins.

Since Robert Brown's first observations of random walks by pollen particles suspended in solution, the concept of diffusion has been subject to countless theoretical and experimental studies in diverse fields from finance and social sciences, to physics and biology. Diffusive transport of macromolecules in cells is intimately linked to essential cellular functions including nutrient uptake, signal transduction, gene expression, as well as DNA replication and repair. Advancement in experimental techniques has allowed precise measurements of these diffusion processes. Mathematical and physical descriptions and computer simulations have been applied to model complicated biological systems in which anomalous diffusion, in addition to simple Brownian motion, was observed. The purpose of this review is to provide an overview of the major physical models of anomalous diffusion and corresponding experimental evidence on the target search problem faced by DNA-binding proteins, with an emphasis on DNA repair proteins and the role of anomalous diffusion in DNA target recognition.

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.

Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the ß-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of ß-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance.