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Abstract CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix–loop–helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH’s conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9–sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure–dynamic–function relationship governing DNA target discrimination by Cas9. 

Abstract DNA methylation at cytosine bases (5-methylcytosine, 5mC) is a heritable epigenetic mark regulating gene expression. While enzymes that metabolize 5mC are well-characterized, endogenous signaling molecules that regulate DNA methylation machinery have not been described. We report that physiological nitric oxide (NO) concentrations reversibly inhibit the DNA demethylases TET and ALKBH2 by binding to the mononuclear non-heme iron atom forming a dinitrosyliron complex (DNIC) and preventing cosubstrates from binding. In cancer cells treated with exogenous NO, or endogenously synthesizing NO, 5mC and 5-hydroxymethylcytosine (5hmC) increase, with no changes in DNA methyltransferase activity. 5mC is also significantly increased in NO-producing patient-derived xenograft tumors from mice. Genome-wide methylome analysis of cells chronically treated with NO (10 days) shows enrichment of 5mC and 5hmC at gene-regulatory loci, correlating with altered expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a unique epigenetic role for NO. 

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3 CO TP) is synthesized and site-specifically incorporated into large RNAs by in vitro transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy by SDSL of a 419-nucleotide ribonuclease P (RNase P) RNA from Bacillus stearothermophilus under non-denaturing conditions. The effects of site-directed UBP incorporation and subsequent spin labeling on the global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, Sedimentation Velocity Analytical Ultracentrifugation and enzymatic assay. Continuous-Wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron–Electron Double Resonance measurements yield an inter-spin distance distribution that agrees with the crystal structure. The labeling strategy as presented overcomes the size constraint of RNA labeling, opening new avenues of spin labeling and EPR spectroscopy for investigating the structure and dynamics of large RNAs. 

We report a rare example of a mixed-valence iron compound with an FeNNFe core, which gives insight into the structural, spectroscopic, and magnetic influences of single-electron reductions and oxidations. In the new compound, the odd electron is localized as judged from Mössbauer spectra at 80 K and infrared spectra at room temperature, and the backbonding into the N 2 unit is intermediate between diiron( i ) and diiron(0) congeners. Magnetic susceptibility and relaxation studies on the series of FeNNFe compounds show significant magnetic anisotropy, but through-barrier pathways enable fairly rapid magnetic relaxation. 

Cas12a is an RNA‐guided DNA endonuclease of the type V‐A CRISPR‐Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity inStreptococcus pyogenes(Spy) Cas9. Here, we examined a BH variant of Cas12a fromFrancisella novicida(FnoCas12a^KD2P) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage (cis‐activity), FnoCas12a^KD2Paccumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12a^KD2Palso possess reducedtrans‐single‐stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in bothcis‐andtrans‐cleavages and show its conserved role in target discrimination among Cas nucleases.