EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding-degradation coordination of an exonuclease. Here, using a single-molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA–DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2-mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4-bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate-limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.

中文翻译：

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EXD2 核酸外切酶的离散 RNA-DNA 杂交切割精确定位了两个限速步骤

EXD2 是最近发现的一种核酸外切酶，可切割双链 (ds) 形式的 RNA 和 DNA。因此，它可作为研究外切核糖核酸酶和外切脱氧核糖核酸酶活性之间的相似性和差异以及了解外切核酸酶的核酸（NA）解旋-降解协调的模型系统。在这里，使用单分子荧光共振能量转移（smFRET）方法，我们表明，尽管与两种底物稳定结合，EXD2几乎不切割双链DNA，但对RNA-DNA杂交体显示出核糖核酸外切酶和脱氧核糖核酸外切酶活性，并且对RNA具有切割偏好。出乎意料的是，EXD2介导的杂合裂解以离散的逐步模式进行，其中突然的4-bp双链体解旋增量和随后的停留构成了完整的水解循环。EXD2相对较弱的外切脱氧核糖核酸酶活性部分源于频繁的杂交倒带。重要的是，动力学分析和不同条件下停留时间的比较揭示了杂交解旋和核苷酸切除的两个限速步骤。总的来说，我们的研究结果有助于更好地理解 EXD2 的细胞功能，并且双链体解旋和核酸外切降解之间的循环耦合可能可以推广到其他核酸外切酶。