Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

The heterochromatin spreading reaction is a central contributor to the formation of gene-repressive structures, which are re-established with high positional precision, or fidelity, following replication. How the spreading reaction contributes to this fidelity is not clear. To resolve the origins of stable inheritance of repression, we probed the intrinsic character of spreading events in fission yeast using a system that quantitatively describes the spreading reaction in live single cells. We show that spreading triggered by noncoding RNA-nucleated elements is stochastic, multimodal, and fluctuates dynamically across time. This lack of stability correlates with high histone turnover. At the mating type locus, this unstable behavior is restrained by an accessory cis-acting element REIII, which represses histone turnover. Further, REIII safeguards epigenetic memory against environmental perturbations. Our results suggest that the most prevalent type of spreading, driven by noncoding RNA-nucleators, is epigenetically unstable and requires collaboration with accessory elements to achieve high fidelity.

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeqnuclease digestion and deep sequencing-a massively parallel platform that measures cleavage kinetics and captures the timeresolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a-reported as a more specific nuclease in cells-has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.A ll DNA maintenance processes require nucleases, which enzymatically cleave the phosphodiester bonds in nucleic acids. Exo1, a member of the Rad2 family of nucleases, participates in DNA mismatch repair (MMR), double-strand break (DSB) repair, nucleotide excision repair (NER), and telomere maintenance (1-3). Exo1 is the only nuclease implicated in MMR, where its 5ʹ to 3ʹ exonuclease activity is used to remove long tracts of mismatch-containing single-stranded DNA (ssDNA) (2, 4-7). In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8, 9). Exo1 also promotes DSB repair via homologous recombination (HR) by processing the free DNA ends to generate kilobase-length ssDNA resection products (1, 10-12). The resulting ssDNA is paired with a homologous DNA sequence located on a sister chromatid, and the missing genetic information is then restored via DNA synthesis. The central role of Exo1 in DNA repair is highlighted by the large set of genetic interactions between Exo1 and nearly all other DNA maintenance and metabolism pathways (13).Exo1 generates long tracts of ssDNA in both MMR and DSB repair (3). This ssDNA is rapidly bound by replication protein A (RPA), a ubiquitous heterotrimeric protein that participates in all DNA transactions that generate ssDNA intermediates (14). RPA protects the ssDNA from degradation, participates in DNA damage response signaling, and acts as a loading platform for downstream DSB repair proteins (15-17). RPA also coordinates DNA resection by removing secondary ssDNA structures and by modulating the Bloom syndrome, RecQ helicase-like (BLM)/ DNA2-and Ex...

Precision genome editing technologies have transformed modern biology. These technologies have arisen from the redirection of natural biological machinery, such as bacteriophage lambda proteins for recombineering and CRISPR nucleases for eliciting site-specific double-strand breaks. Less well-known is a widely distributed class of bacterial retroelements, retrons, that employ specialized reverse transcriptases to produce noncoding intracellular DNAs. Retrons’ natural function and mechanism of genetic transmission have remained enigmatic. However, recent studies have harnessed their ability to produce DNA in situ for genome editing and evolution. This review describes retron biology and function in both natural and synthetic contexts. We also highlight areas that require further study to advance retron-based precision genome editing platforms.