Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.

Indels (insertions and deletions) are the second most common form of genetic variations in the eukaryotic genomes and are responsible for a multitude of genetic diseases. Despite its significance, detailed molecular mechanisms for indel generation are still unclear. Here we examined 2,656,597 small human and mouse germline indels, 16,742 human somatic indels, 10,599 large human insertions, and 5,822 large chimpanzee insertions and systematically analyzed the patterns of DNA cleavage intensities in the 200 base pair regions surrounding these indels. Our results show that DNA cleavage intensities close to the start and end points of indels are significantly lower than other regions, for both small human germline and somatic indels and also for mouse small indels. Compared to small indels, the patterns of DNA cleavage intensity around large indels are more complex, and there are two low intensity regions near each end of the indels that are approximately 13 bp apart from each other. Detailed analyses of a subset of indels show that there is slight difference in cleavage intensity distribution between insertion indels and deletion indels that could be contributed by their respective enrichment of different repetitive elements. These results will provide new insight into indel generation mechanisms.

Environmental and endogenous toxins are considered to increase the risk of dopaminergic neurodegeneration. Parkinson's disease is a neurological disorder occurring due to the death of dopaminergic neurons in substantia nigra. The present study investigated the effect of parkinsonian neurotoxins salsolinol and rotenone on plasmid and genomic DNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable 'clamped' Cas12a-DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3' CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3' CRISPR RNA to downstream DNA.

The preimplantation period of embryogenesis is crucial during mammalian ontogenesis. During this period, the mitotic cycles are initiated, the embryonic genome is activated, and the primary differentiation of embryonic cells occurs. All cellular abnormalities occurring in this period are the primary cause of fetal developmental disorders. DNA damage is a serious cause of developmental failure. In the context of DNA damage response on the cellular level, we analyzed the course of embryogenesis and phenotypic changes during the cleavage of a preimplantation embryo. Our results document that DNA damage induced before the resumption of DNA synthesis in a zygote can significantly affect the preimplantation development of the embryo. This developmental ability is related to the level of the DNA damage. We showed that one-cell embryos can correct the first cleavage cycle despite low DNA damage and incomplete replication. It seems that the phenomenon creates a predisposition to a segregation disorder of condensed chromatin that results in the formation of micronuclei in the developmental stages following the first cleavage. We conclude that zygote tolerates a certain degree of DNA damage and considers its priority to complete the first cleavage stage and continue embryogenesis as far as possible.

EGFR may induce DNA degradation. This activity had not been previously described as an EGRF function. To confirm this unexpected activity, testing of EGFR in the presence of ATP and either 5A, 5C, 5G, 5T, or 5U oligonucleotides was performed. HPLC-MS analysis demonstrated that 5A and 5U levels significantly decreased in the presence of EGFR. Furthermore, fragments 4A and 4U were produced in 5A+EGFR+ATP and in 5U+EGFR+ATP reaction mixtures, respectively, but not in EGFR-negative controls. Degradation of Poly(A), Poly(C), Poly(G), Poly(I), Poly(T), and Poly(U) oligomers in the presence of EGFR and ATP correlated with the lower ability of reaction products to pair with complementary oligonucleotides. Gel electrophoresis showed that breakdown products migrated more quickly than controls, especially after addition of paired (complementary) oligomers, Poly(A) and Poly(U). Furthermore, λ DNA reaction products also migrated more quickly after incubation with EGFR. The results suggest that EGFR can induce breakage of certain types of nucleotide phosphodiester bonds, especially within the A residues of DNA or U residues of RNA, to induce DNA or RNA decomposition, respectively. This activity may be important in EGRF signaling, DNA degradation, or repair in normal or cancer cell activities.

Homing endonucleases are used in a wide range of biotechnological applications including gene editing, in gene drive systems, and for the modification of DNA structures, arrays, and prodrugs. However, controlling nuclease activity and sequence specificity remain key challenges when developing new tools. Here a photoresponsive homing endonuclease was engineered for optical control of DNA cleavage by partitioning DNA binding and nuclease domains of the monomeric homing endonuclease I-TevI into independent polypeptide chains. Use of the Aureochrome1a light-oxygen-voltage domain delivered control of dimerization with light. Illumination reduced the concentration needed to achieve 50% cleavage of the homing target site by 6-fold when compared to the dark state, resulting in an up to 9-fold difference in final yields between cleavage products. I-TevI nucleases with and without a native I-TevI zinc finger motif displayed different nuclease activity and sequence preference impacting the promiscuity of the nuclease domain. By harnessing an alternative DNA binding domain, target preference was reprogrammed only when the nuclease lacked the I-TevI zinc finger motif. This work establishes a first-generation photoresponsive platform for spatiotemporal activation of DNA cleavage.

Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.

Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a β- or β,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both β- and β,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring.

Approaches developed for sequencing DNA with detection by mass spectrometry use strategies that deviate from the Sanger-type methods. Procedures demonstrated so far used the sequence specificity of RNA endonucleases, as unfortunately equivalent enzymes for DNA do not exist and therefore require transcription of DNA into RNA prior to fragmentation. We have developed a novel, rapid and accurate concept for DNA sequencing using mass spectrometry and RNA/DNA chimeras and applied it to sequence mitochondrial DNA. Our method is based on the preparation of a chimeric RNA/DNA with a DNA polymerase that also incorporates ribonucleotides. Sequencing is carried out with one ribonucleotide (ATP, CTP or GTP) and the other three nucleotides in their deoxyribo-form. The product is treated with alkali, which cleaves 3' of all ribonucleotides to form a terminal 3' phosphate. Conditions have been streamlined so that molecular, biological and alkali cleavage conditions are compatible with matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometric analysis. Fragment analysis by MALDI MS provides a sequence-specific fingerprint, which allows the identification of differences between a reference and another sequence. Due to the mass profile, the position and kind of the mutation can be assigned. These differences between signatures are indicative of known, unidentified, rare and private mutations. This novel DNA sequencing protocol was applied to sequence the hypervariable region 1 (HV1) of mitochondrial DNA in 22 individuals.

CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions. Substrate requirements for the RNA-independent activity vary. FnoCas9 preferentially nicks double-stranded plasmid, SpyCas9 degrades single-stranded plasmid, and FnoCas12a cleaves both substrates. These observations suggest that the identities and levels of intracellular metals, along with the Cas9/Cas12a ortholog employed, could have significant impacts in genome editing applications.

Deoxyribonucleic acid (DNA) topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. The camptothecin (CPT) Top1 (topoisomerase I) inhibitors exert their anticancer activity by reversibly trapping Top1-DNA cleavage complexes (Top1cc's) and inducing replication-associated DNA double-strand breaks (DSBs). In this paper, we propose a new mechanism by which cells avoid Top1-induced replication-dependent DNA damage. We show that the structure-specific endonuclease Mus81-Eme1 is responsible for generating DSBs in response to Top1 inhibition and for allowing cell survival. We provide evidence that Mus81 cleaves replication forks rather than excises Top1cc's. DNA combing demonstrated that Mus81 also allows efficient replication fork progression after CPT treatment. We propose that Mus81 cleaves stalled replication forks, which allows dissipation of the excessive supercoiling resulting from Top1 inhibition, spontaneous reversal of Top1cc, and replication fork progression.

A series of selenylated-oxadiazoles were prepared and their interaction with DNA was investigated. The photophysical studies showed that all the selenylated compounds presented absorption between 270 and 329 nm, assigned to combined n→π* and π→π* transitions, and an intense blue emission (325-380 nm) with quantum yield in the range of Φ F = 0.1-0.4. DFT and TD-DFT calculations were also performed to study the likely geometry and the excited state of these compounds. Electrochemical studies revealed the ionization potential energies (-5.13 to -6.01 eV) and electron affinity energies (-2.25 to -2.83 eV), depending directly on the electronic effect (electron-donating or electron-withdrawing) of the substituent attached to the product. Finally, the UV-Vis DNA interaction experiments indicated that the compounds can interact with the DNA molecule due to intercalation, except for 3g (which interacted via electrostatic interaction). Plasmid cleavage assay presented positive results only for 3f that presented the strongest interaction results. These results made the tested selenylated-oxadiazoles as suitable structures for the development of drugs and the design of structurally-related therapeutics.

The design of molecules that damage a selected DNA sequence provides a formidable opportunity for basic and applied biology. For example, such molecules offer new prospects for controlled manipulation of the genome. The conjugation of DNA-code reading molecules such as polyamides to reagents that induce DNA damages provides an approach to reach this goal. In this work, we showed that a bipyridine conjugate of polyamides was able to induce sequence-specific DNA breaks in cells. We synthesized compounds based on two polyamide parts linked to bipyridine at different positions. Bipyridine conjugates of polyamides were found to have a high affinity for the DNA target and one of them produced a specific and high-yield cleavage in vitro and in cultured cells. The bipyridine conjugate studied here, also presents cell penetrating properties since it is active when directly added to cell culture medium. Harnessing DNA damaging molecules such as bipyridine to predetermined genomic sites, as achieved here, provides an attractive strategy for targeted genome modification and DNA repair studies.

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as 'topological poisons of topoisomerase I'.

C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to single-nucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases.

CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9.

The initiation of embryonic gene expression in ascidian embryos appears to be tightly regulated by the number of DNA replication cycles. DNA methylation is thought to contribute to the clock mechanism that counts the rounds of DNA replication. We used mass spectrometry and whole genome bisulfite sequencing to characterize DNA methylation changes that occur in early developmental stages of the ascidian, Ciona intestinalis. We found that global DNA methylation in early Ciona development was static, and a base-wise comparison between the genomes of consecutive developmental stages found no DNA demethylation that was related to zygotic gene activation. Additionally, 5hmC was hardly detected by mass spectrometry in the developmental samples, suggesting a lack of demethylation mediated by ten eleven translocation (TET) methylcytosine dioxygenase in C. intestinalis. We conclude that DNA methylation is not involved in regulating DNA replication-dependent transcriptional activation.