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1. Unidirectional trans -cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3′ toehold

   https://doi.org/10.1093/nar/gkad715  

   Mohammad, Noor; Talton, Logan; Hetzler, Zach; Gongireddy, Megha; Wei, Qingshan (August 2023, Nucleic Acids Research)

   Abstract CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn’t cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3′ overhang dsDNA substrates at least 3 times faster than 5′ overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule. 

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2. Assembly mechanism of the inflammasome sensor AIM2 revealed by single molecule analysis

   https://doi.org/10.1038/s41467-023-43691-4  

   Sharma, Meenakshi; de Alba, Eva (December 2023, Nature Communications)

   Abstract Pathogenic dsDNA prompts AIM2 assembly leading to the formation of the inflammasome, a multimeric complex that triggers the inflammatory response. The recognition of foreign dsDNA involves AIM2 self-assembly concomitant with dsDNA binding. However, we lack mechanistic and kinetic information on the formation and propagation of the assembly, which can shed light on innate immunity’s time response and specificity. Combining optical traps and confocal fluorescence microscopy, we determine here the association and dissociation rates of the AIM2-DNA complex at the single molecule level. We identify distinct mechanisms for oligomer growth via the binding of incoming AIM2 molecules to adjacent dsDNA or direct interaction with bound AIM2 assemblies, resembling primary and secondary nucleation. Through these mechanisms, the size of AIM2 oligomers can increase fourfold in seconds. Finally, our data indicate that single AIM2 molecules do not diffuse/scan along the DNA, suggesting that oligomerization depends on stochastic encounters with DNA and/or DNA-bound AIM2. 

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3. Filament assembly underpins the double-stranded DNA specificity of AIM2-like receptors

   https://doi.org/10.1093/nar/gkad090  

   Garg, Archit; Stallings, Christina M.; Sohn, Jungsan (March 2023, Nucleic Acids Research)

   Abstract Upon sensing cytosolic- and/or viral double-stranded (ds)DNA, absent-in-melanoma-2 (AIM2)-like-receptors (ALRs) assemble into filamentous signaling platforms to initiate inflammatory responses. The versatile yet critical roles of ALRs in host innate defense are increasingly appreciated; however, the mechanisms by which AIM2 and its related IFI16 specifically recognize dsDNA over other nucleic acids remain poorly understood (i.e. single-stranded (ss)DNA, dsRNA, ssRNA and DNA:RNA hybrid). Here, we find that although AIM2 can interact with various nucleic acids, it preferentially binds to and assembles filaments faster on dsDNA in a duplex length-dependent manner. Moreover, AIM2 oligomers assembled on nucleic acids other than dsDNA not only display less ordered filamentous structures, but also fail to induce the polymerization of downstream ASC. Likewise, although showing broader nucleic acid selectivity than AIM2, IFI16 binds to and oligomerizes most readily on dsDNA in a duplex length-dependent manner. Nevertheless, IFI16 fails to form filaments on single-stranded nucleic acids and does not accelerate the polymerization of ASC regardless of bound nucleic acids. Together, we reveal that filament assembly is integral to nucleic acid distinction by ALRs. 

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4. Mechanism of DNA Intercalation by Chloroquine Provides Insights into Toxicity

   https://doi.org/10.3390/ijms25031410  

   Joshi, Joha; McCauley, Micah J; Morse, Michael; Muccio, Michael R; Kanlong, Joseph G; Rocha, Márcio S; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C (February 2024, International Journal of Molecular Sciences)

   Chloroquine has been used as a potent antimalarial, anticancer drug, and prophylactic. While chloroquine is known to interact with DNA, the details of DNA–ligand interactions have remained unclear. Here we characterize chloroquine–double-stranded DNA binding with four complementary approaches, including optical tweezers, atomic force microscopy, duplex DNA melting measurements, and isothermal titration calorimetry. We show that chloroquine intercalates into double stranded DNA (dsDNA) with a KD ~ 200 µM, and this binding is entropically driven. We propose that chloroquine-induced dsDNA intercalation, which happens in the same concentration range as its observed toxic effects on cells, is responsible for the drug’s cytotoxicity. 

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5. MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

   https://doi.org/10.1038/s41586-023-06889-6  

   Cho, Min-Guk; Kumar, Rashmi J; Lin, Chien-Chu; Boyer, Joshua A; Shahir, Jamshaid A; Fagan-Solis, Katerina; Simpson, Dennis A; Fan, Cheng; Foster, Christine E; Goddard, Anna M; et al (January 2024, Nature)

   Abstract Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS–STING-mediated signalling and tumour suppression^1–3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)^4–10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11–RAD50–NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1–RIPK3–MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation ofZBP1in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis. 

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