Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance

Abstract

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1

“…We previously used ubiquitinated H3 peptide analogs with an unnatural isopeptide mimetic structure for investigating the effect of this molecule on Dnmt1 enzymatic activity . In this research, in order to confirm the reliability of the analogs, we synthesized ubiquitinated H3 peptides with a native isopeptide structure at Lys18 or/and 23 position(s), as shown in Scheme .…”

“…Analogs with the structures shown in Figure B showed a much enhanced Dnmt1 activity. The disulfide analogs, shown in Figure C, cannot be used in this measurement, because the experiment was performed under reduced conditions in the presence of DTT, but isothermal calorimetry and X‐ray crystal experiments showed a tight binding of the analogs to the domain of Dnmt1 with the similar order in enzymatic enhancement at the ubiquitinated positions under non‐reducing conditions . Compared with these structures, the native isopeptide structure might be more flexible than the analogs.…”

“…For Dnmt1, in which N‐terminal 290 amino acids are truncated, the expression plasmid using pFASTBAC system (Invitrogen) was prepared, and expressed in insect cells . Ubiqutinated histone H3 peptides and their analogs were renatured …”

“…Methylation activity measurements using radioisotopes were performed as described . Hemi‐methylated DNA, which is comprised of 42bp and 12 methylated CpG, were used as substrates .…”

“…In the cell division process, newly synthesized DNA is hemi‐methylated and is converted to its fully methylated form mainly by DNA methyltransferase 1 (Dnmt1) to maintain the methylation pattern . The ubiquitination of H3 at Lys18 or/and 23 was reported to regulate the maintenance DNA methylation by Dnmt1 …”