UHRF1 plays multiple roles in regulating DNMT1-mediated DNA methylation maintenance during DNA replication. The UHRF1 C-terminal RING finger functions as an ubiquitin E3 ligase to establish histone H3 ubiquitination at Lys18 and/or Lys23, which is subsequently recognized by DNMT1 to promote its localization onto replication foci. Here, we present the crystal structure of DNMT1 RFTS domain in complex with ubiquitin and highlight a unique ubiquitin binding mode for the RFTS domain. We provide evidence that UHRF1 N-terminal ubiquitin-like domain (UBL) also binds directly to DNMT1. Despite sharing a high degree of structural similarity, UHRF1 UBL and ubiquitin bind to DNMT1 in a very distinct fashion and exert different impacts on DNMT1 enzymatic activity. We further show that the UHRF1 UBL-mediated interaction between UHRF1 and DNMT1, and the binding of DNMT1 to ubiquitinated histone H3 that is catalyzed by UHRF1 RING domain are critical for the proper subnuclear localization of DNMT1 and maintenance of DNA methylation. Collectively, our study adds another layer of complexity to the regulatory mechanism of DNMT1 activation by UHRF1 and supports that individual domains of UHRF1 participate and act in concert to maintain DNA methylation patterns.
Background:The typical two-cysteine peroxiredoxin Ahp1 catalyzes the decomposition of alkyl hydroperoxides. Results: Ahp1 possesses a peroxidatic Cys-62 after resolving Cys-31 and an A-type dimer interface that contributes to the positive cooperativity of hydroperoxide binding. Conclusion: Ahp1 defines a novel group of thioredoxin-dependent peroxidases. Significance: Provided is the first structural snapshot of electron transfer from thioredoxin to a thioredoxin-like protein.
BackgroundThe carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: α-, β-, γ-, δ-, ε-, ζ- CAs, with β-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of β-CA Nce103/YNL036W.ResultsWe determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 Å resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable.ConclusionThe quaternary structure of Nce103 resembles the typical plant type β-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.
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