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

The Proliferating Cell Nuclear Antigen-associated factor p15 is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15 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 p15 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 p15 monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15 is monomeric, intrinsically disordered, binds to PCNA as non-ubiquitinated p15 does, but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15 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 p15 is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15 directly interacts with PCNA and recruits Dnmt1for maintenance of DNA methylation during replication.