Activation of DNMT1 by Chemically Synthesized Dual-Monoubiquitinated PAF15 Protein.

Abstract

Replication-coupled DNA methylation maintenance by DNA methyltransferase 1 (DNMT1) is mediated by dual-monoubiquitinated PCNA-associated factor 15 (PAF15Ub2). However, the molecular mechanism of DNMT1 activation by PAF15Ub2 remains unclear. Here, taking advantage of chemical protein synthesis, we prepare PAF15 protein bearing dual-monoubiquitination through native amide linkages and determine the active conformation of DNMT1 complexed with the synthetic PAF15Ub2 and hemimethylated DNA by cryogenic electron microscopy (cryo-EM). With an optimized convergent synthetic route, the 262 amino-acid protein PAF15Ub2 was assembled from six synthetic peptide segments, including two branched peptides. By employing a one-pot ligation technique and deselenization chemistry, we obtained the native sequence of PAF15Ub2 in high purity and good yield. The chemically synthesized PAF15Ub2 was shown to bind to DNMT1 and stimulate its methyltransferase activity in biochemical assays. Cryo-EM analysis of DNMT1 in complex with PAF15Ub2 and hemimethylated DNA revealed that DNMT1 adopts an active conformation in which the RFTS domain is released from the catalytic domain, thereby relieving the autoinhibition.

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