Decoding the base flipping mechanism of the SET- and RING-associated (SRA) domain of the epigenetic UHRF1 protein.

Ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays a pivotal role in replicating DNA methylation patterns during cell division. Acting as a DNA reader, UHRF1, via its SET- and RING-associated (SRA) domain, recognizes hemi-methylated (HM) CpG sites and flips 5-methylcytosine (5mC) nucleobases. This flipping triggers DNA methyltransferase 1 (DNMT1) recruitment to methylate cytosine in the complementary strand. To investigate the SRA-induced base-flipping mechanism, we introduced thienoguanosine (thG), a fluorescent guanosine analogue, at four positions in HM and non-methylated duplexes. The interactions of these labelled duplexes with wild-type SRA and a G448D mutant (incapable of base-flipping) were monitored using a combination of stopped-flow fluorescence measurements, molecular dynamics simulations, and quantum mechanical calculations. We show that 5mC and C residues are flipped with similar rate constants. However, while C residues rapidly revert to their original state, enabling SRA to continue reading or dissociate, SRA complexes with flipped 5mC undergo a slow conformational rearrangement, leading to the final conformation crucial for DNMT1 recruitment. Taken together, our findings suggest that base flipping is used to discriminate 5mC from C residues, while the ensuing conformational rearrangement drives DNMT1 recruitment.