Double Monoubiquitination Modifies the Molecular Recognition Properties of p15^PAF Promoting Binding to the Reader Module of Dnmt1

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 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, 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.

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation

“…These structural features are also consistent with the fact that early replicating domains contain a much higher degree of DNA methylation at which time PAF15Ub2 is predominantly recruiting DNMT1. We also note that, during the revision of our manuscript, a recent study has also shown that full-length hPAF15Ub2 binds DNMT1 in vitro 49 .…”

Human PCNA Structure, Function and Interactions

“…The monoubiquitination of lysine residues 15 and 24 by UHRF1 in the N-terminal tail of p15 appears to be extremely important. Recently, an extensive structural and conformational characterization of doubly monoubiquitinated p15 showed that it remains disordered and binds PCNA in the same way as non-ubiquitinated p15, but binds DNA with a 5-fold reduced affinity [55], which supports the previously proposed hypothesis of p15 modulating the velocity with which PCNA slides along DNA [54]. Moreover, calorimetry experiments revealed that the double monoubiquitination mark of p15 is recognized by the RFTS domain of DNA methyl transferase 1, indicating a role of p15 in the regulation of DNA methylation maintenance.…”

Emerging Roles and Research Tools of Atypical Ubiquitination

“…Moreover, monoubiquitination and multi‐monoubiquitination have been reported to regulate protein–protein interactions. For example, doubly monoubiquitinated p15PAF interacts directly with PCNA and facilitates the recruitment of Dnmt1 to maintain DNA methylation during DNA replication . Similarly, Zhang et al.…”