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DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B–3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

RNase R is a conserved exoribonuclease in the RNase II family that primarily participates in RNA decay in all kingdoms of life. RNase R degrades duplex RNA with a 3′ overhang, suggesting that it has RNA unwinding activity in addition to its 3′-to-5′ exoribonuclease activity. However, how RNase R coordinates RNA binding with unwinding to degrade RNA remains elusive. Here, we report the crystal structure of a truncated form of Escherichia coli RNase R (residues 87–725) at a resolution of 1.85 Å. Structural comparisons with other RNase II family proteins reveal two open RNA-binding channels in RNase R and suggest a tri-helix ‘wedge’ region in the RNB domain that may induce RNA unwinding. We constructed two tri-helix wedge mutants and they indeed lost their RNA unwinding but not RNA binding or degrading activities. Our results suggest that the duplex RNA with an overhang is bound in the two RNA-binding channels in RNase R. The 3′ overhang is threaded into the active site and the duplex RNA is unwound upon reaching the wedge region during RNA degradation. Thus, RNase R is a proficient enzyme, capable of concurrently binding, unwinding and degrading structured RNA in a highly processive manner during RNA decay.

Human Tudor-SN is involved in the degradation of hyper-edited inosine-containing microRNA precursors, thus linking the pathways of RNA interference and editing. Tudor-SN contains four tandem repeats of staphylococcal nuclease-like domains (SN1–SN4) followed by a tudor and C-terminal SN domain (SN5). Here, we showed that Tudor-SN requires tandem repeats of SN domains for its RNA binding and cleavage activity. The crystal structure of a 64-kD truncated form of human Tudor-SN further shows that the four domains, SN3, SN4, tudor and SN5, assemble into a crescent-shaped structure. A concave basic surface formed jointly by SN3 and SN4 domains is likely involved in RNA binding, where citrate ions are bound at the putative RNase active sites. Additional modeling studies provide a structural basis for Tudor-SN's preference in cleaving RNA containing multiple I·U wobble-paired sequences. Collectively, these results suggest that tandem repeats of SN domains in Tudor-SN function as a clamp to capture RNA substrates.

Exonucleases are key enzymes in the maintenance of genome stability, processing of immature RNA precursors and degradation of unnecessary nucleic acids. However, it remains unclear how exonucleases digest nucleic acids to generate correct end products for next-step processing. Here we show how the exonuclease RNase T stops its trimming precisely. The crystal structures of RNase T in complex with a stem-loop DNA, a GG dinucleotide and single-stranded DNA with different 3'-end sequences demonstrate why a duplex with a short 3'-overhang, a dinucleotide and a ssDNA with a 3'-end C cannot be further digested by RNase T. Several hydrophobic residues in RNase T change their conformation upon substrate binding and induce an active or inactive conformation in the active site that construct a precise machine to determine which substrate should be digested based on its sequence, length and structure. These studies thus provide mechanistic insights into how RNase T prevents over digestion of its various substrates, and the results can be extrapolated to the thousands of members of the DEDDh family of exonucleases.