Exploiting Substrate Promiscuity To Develop Activity-Based Probes for Ten-Eleven Translocation Family Enzymes
journal contributionposted on 06.12.2018, 00:00 by Uday Ghanty, Jamie E. DeNizio, Monica Yun Liu, Rahul M. Kohli
Ten-eleven translocation (TET) enzymes catalyze repeated oxidations of 5-methylcytosine in genomic DNA. Because of the challenges of tracking reactivity within a complex DNA substrate, chemical tools to probe TET activity are limited, despite these enzyme’s crucial role in epigenetic regulation. Here, building on precedents from related Fe(II)/α-ketoglutarate-dependent dioxygenases, we show that TET enzymes can promiscuously act upon cytosine bases with unnatural 5-position modifications. Oxidation of 5-vinylcytosine (vC) in DNA results in the predominant formation of a 5-formylmethylcytosine product that can be efficiently labeled to provide an end-point read-out for TET activity. The reaction with 5-ethynylcytosine (eyC), moreover, results in the formation of a high-energy ketene intermediate that can selectively trap any active TET isoform as a covalent enzyme–DNA complex, even in the complex milieu of a total cell lysate. Exploiting substrate promiscuity therefore offers a new and needed means to directly track TET activity in vitro or in vivo.
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5- methylcytosine5- ethynylcytosineend-point read-outcell lysateTET activityDNA substrateprobe TET activityTen-Eleven Translocation Family Enzymes Ten-eleven translocation5- vinylcytosinetrack TET activitychemical toolsgenomic DNATET enzymes5- position modificationsExploiting substrate promiscuityExploiting Substrate PromiscuityActivity-Based Probespromiscuously actDNA resultsenzymes catalyze5- formylmethylcytosine productTET isoformcytosine bases