Replication of the ∼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development.
DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.
DNA methyltransferase DNMT3B plays an essential role in establishment of DNA methylation during embryogenesis. Mutations of DNMT3B are associated with human diseases, notably the immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome. How ICF mutations affect DNMT3B activity is not fully understood. Here we report the homo-oligomeric structure of DNMT3B methyltransferase domain, providing insight into DNMT3B-mediated DNA methylation in embryonic stem cells where the functional regulator DNMT3L is dispensable. The interplay between one of the oligomer interfaces (FF interface) and the catalytic loop renders DNMT3B homo-oligomer a conformation and activity distinct from the DNMT3B-DNMT3L heterotetramer, and a greater vulnerability to certain ICF mutations. Biochemical and cellular analyses further reveal that the ICF mutations of FF interface impair the DNA binding and heterochromatin targeting of DNMT3B, leading to reduced DNA methylation in cells. Together, this study provides a mechanistic understanding of DNMT3B-mediated DNA methylation and its dysregulation in disease.
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