Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1 González-Magaña, Amaia Ibáñez de Opakua, Alain Merino, Nekane Monteiro, Hugo Diercks, Tammo Murciano Calles, Javier Luque Fernández, Irene Bernadó, Pau Cordeiro, Tiago De Biasio, Alfredo Blanco, Francisco J. The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its Nterminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication. 2019-11-11T11:55:03Z 2019-11-11T11:55:03Z 2019-09-03 journal article Amaia González-Magaña, Alain Ibáñez de Opakua, Nekane Merino, Hugo Monteiro, Tammo Diercks, Javier Murciano-Calles, Irene Luque, Pau Bernadó, Tiago N. Cordeiro, Alfredo De Biasio, and Francisco J. Blanco ACS Chem. Biol. 2019, 14, 2315−2326 [DOI: 10.1021/acschembio.9b00679] http://hdl.handle.net/10481/57814 10.1021/acschembio.9b00679 eng http://creativecommons.org/licenses/by-nc-nd/3.0/es/ open access Atribución-NoComercial-SinDerivadas 3.0 España American Chemical Society