Biology subjects

Rapoport, T.

Publications and source records attributed to Rapoport, T..

3 recordsLinked to original sources

bioRxiv2026

Molecular Logic of DNMT3A1 Recruitment: Resolving Multivalency at the Chromatin Interface

Histone modifications correlate with DNA methylation, but the underlying mechanisms remain unclear. The DNA methyltransferase DNMT3A1 engages nucleosomes through multivalent interactions with linker DNA, histone H3 tails, the acidic patch, and ubiquitinated H2A. Using binding assays, kinetic analyses, and nanopore sequencing of engineered nucleosomes, we show that DNMT3A1 binding reflects avidity, limiting the impact of individual contacts. Accordingly, disrupting single interactions minimally affects overall affinity, whereas isolated domains and truncations remain modification-sensitive. Although histone modifications have little effect on kcat/Km for linker methylation, disrupting ADD-H3K4me0 interactions redistributes methylation away from the nucleosome core particle. Nucleosome competition assays reveal that H3K4me0 and H3K36me2 promote selective linker methylation, whereas H3K27me3 and PRC2/EZH2 have no effect. Notably, despite strong UDR-dependent binding to H2AK119ub1-modified nucleosomes, this mark fails to enhance methylation over an unmodified competitor. We propose a model of commitment to catalysis to reconcile weak kinetic differences with strong substrate selectivity. These findings highlight avidity and commitment in governing DNMT3A1 nucleosome recognition and DNA methylation specificity.

molecular biology↗

bioRxiv2025

The deubiquitinating enzyme Otu1 releases substrates from the conserved initiation complex of the Cdc48/p97 ATPase for proteasomal degradation

Many eukaryotic proteins are modified with a polyubiquitin chain and then recruited to either the Cdc48 ATPase (p97 or VCP in mammals) or the 26S proteasome by conserved cofactors. They can then shuttle between the Cdc48 ATPase and the 26S proteasome before being degraded. How substrates avoid being trapped on the Cdc48 ATPase complex is incompletely understood, as they can undergo repeated cycles of translocation through the ATPase pore. Here, we show that the deubiquitinating enzyme (DUB) Otu1 (Yod1 in mammals) can break this futile cycle. Otu1 trims the ubiquitin chain of the substrate before its translocation through the Cdc48 pore is initiated, allowing transfer to the proteasome and subsequent degradation. A cryo-EM structure shows that the mammalian homolog Yod1 binds to p97 simultaneously with other Cdc48 cofactors. As in the yeast system, polypeptide translocation through the ATPase pore is initiated by the unfolding of a ubiquitin molecule, suggesting that the mechanism of substrate processing is conserved in all eukaryotes.

biochemistry↗

bioRxiv2024

Initiation of ERAD by the bifunctional complex of Mnl1 mannosidase and protein disulfide isomerase

Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). In S. cerevisiae, the glycan of these proteins is trimmed by the luminal mannosidase Mnl1 (Htm1) to generate a signal that triggers degradation. Curiously, Mnl1 is permanently associated with protein disulfide isomerase (Pdi1). Here, we have used cryo- electron microscopy, biochemical, and in vivo experiments to clarify how this complex initiates ERAD. The Mnl1-Pdi1 complex first de-mannosylates misfolded, globular proteins that are recognized through a C-terminal domain (CTD) of Mnl1; Pdi1 causes the CTD to ignore completely unfolded polypeptides. The disulfides of these globular proteins are then reduced by the Pdi1 component of the complex, generating unfolded polypeptides that can be translocated across the membrane. Mnl1 blocks the canonical oxidative function of Pdi1, but allows it to function as the elusive disulfide reductase in ERAD.

cell biology↗