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Rafalski, D.

Publications and source records attributed to Rafalski, D..

2 recordsLinked to original sources

Csx15 is a broad specificity ring nuclease bridging CRISPR-Cas and CBASS cyclic nucleotide signaling systems

Prokaryotic antiviral type III CRISPR-Cas and CBASS systems use cyclic nucleotides as second messengers to activate diverse effector proteins. Whereas CBASS signaling typically triggers abortive infection, type III CRISPR-Cas signaling is tightly regulated through controlled signal production and degradation. Here, we define the molecular mechanism of the CRISPR-associated Csx15 protein family and demonstrate that Csx15 functions as a broad specificity ring nuclease capable of degrading cyclic nucleotide signals from both CRISPR-Cas and CBASS pathways. Structural and biochemical analyses reveal that Csx15 adopts diverse oligomeric states and undergoes ligand-induced conformational transitions that promote filament assembly required for catalytic activity, analogous to filament-forming SAVED-domain effectors. Finally, we provide evidence that Csx15 is a close relative of SAVED domains suggesting that Csx15-like proteins may have also contributed to the evolution of CBASS cyclic nucleotide signaling.

biochemistry↗

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic cores of mammalian TET enzymes favor CpGs embedded within bHLH and bZIP transcription factor binding sites, with 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intra-substrate interactions and CpG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germline. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and OCT4, respectively, illuminating TET function in transcriptional responses and pluripotency support. One-Sentence SummaryThe catalytic domains of the enzymes that facilitate passive and drive active DNA demethylation have intrinsic sequence preferences that target DNA demethylation to bHLH and bZIP transcription factor binding sites.

molecular biology↗