Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1
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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.Editorial
American Chemical Society
Date
2019-09-03Referencia bibliográfica
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]
Sponsorship
Spanish Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional (MINECO/FEDER) [CTQ2017-83810-R to F.J.B.]; Labex EpiGenMed, an “Investissements d”avenir’ program [ANR-10-LABX-12-01 to PB]. MOSTMicro [LISBOA-01-0145-FEDER-007660 to T.N.C. and H.M.]. A.G.M. acknowledges Spanish MINECO for predoctoral contract BE-2015-075847, and the CIC bioGUNE acknowledges MINECO for the Severo Ochoa accreditation Sev-2016-0644. The CBS-Montpellier is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), two national infrastructures supported by the French National Research Agency (ANR-10-INSB-04-01 and ANR-10-INSB- 05, respectively).Abstract
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.