Double monoubiquitination modifies the molecular recognition properties of p15 PAF promoting binding to the reader module of Dnmt1

DOI: 10.1021/acschembio.9b00679

Authors: Amaia Gonzalez-magaña (CIC bioGUNE) , Alain Ibáñez De Opakua (CIC bioGUNE) , Nekane Merino (CIC bioGUNE) , Hugo Monteiro (Instituto de Tecnologia Química e Biológica António Xabier, ITQB NOVA) , Tammo Diercks (CIC bioGUNE) , Javier Murciano-calles (Universidad de Granada) , Irene Luque (Universidad de Granada) , Pau Bernadó (INSERM; CNRS; Université Montpellier) , Tiago Cordeiro (Instituto de Tecnologia Química e Biológica António Xabier, ITQB NOVA) , Alfredo De Biasio (University of Leicester) , Francisco J Blanco (CIC bioGUNE; IKERBASQUE, Basque Foundation for Science)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Chemical Biology

State: Published (Approved)
Published: September 2019
Diamond Proposal Number(s): 14707

Abstract: The Proliferating Cell Nuclear Antigen-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 N-terminal 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, binds to PCNA as non-ubiquitinated 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 8 disordered residues, form transient dimers due to the high local effective 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 Dnmt1for maintenance of DNA methylation during replication.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: B21-High Throughput SAXS