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