Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1.

Amaia González-Magaña,Tammo Diercks,Pau Bernadó,Alain Ibáñez De Opakua,Nekane Merino,Javier Murciano-Calles,Irene Luque,Francisco J Blanco,Hugo Monteiro,Alfredo De Biasio,Tiago N Cordeiro

doi:10.1021/acschembio.9b00679

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.

Highlights

Ubiquitination is a multifunctional post-translational modification that may drive the protein through the proteasome degradation pathway, and change the affinity for binding to other biomolecules and, affect protein function in a variety of cellular processes
intrinsically disordered proteins (IDPs) play diverse biological roles and structural disorder is important for the function of proteins that regulate processes often altered in cancer.[32]
One of the functional advantages of IDPs presumably derives from their conformational adaptability, allowing for interactions with different biomolecular partners and for extensive regulation through post-translational modifications at many accessible sites.[33]

Summary

Introduction

Ubiquitination is a multifunctional post-translational modification that may drive the protein through the proteasome degradation pathway, and change the affinity for binding to other biomolecules and, affect protein function in a variety of cellular processes. We present a thorough structural analysis of the p15PAF oncogenic protein monoubiquitinated at two lysine residues via a disulfide bridge that mimics the isopeptide bond. The p15PAF protein (hereafter named p15) is a nuclear 12 kDa polypeptide initially identified as a proliferating cell nuclear antigen (PCNA) binding protein.[2] The DNA sliding clamp PCNA has the shape of a ring and acts as a docking platform for many enzymes that edit DNA.[3] p15 binds to the front face of the PCNA ring as it encircles the DNA and slides along it.[4] Regulatory monoubiquitination at residues K15 and K24 (Figure 1A) selectively occurs on PCNA-bound p15 during the S phase of the cell cycle,[5] when replicative DNA polymerases copy the DNA. The ubiquitination of p15 is mediated by the E3 ligase UHFR1 (ubiquitin-like PHD and RING finger containing domain 1),[6] which monoubiquitinates the histone H3 N-terminal tail at two sites.[7,8] Following

Methods

Results

Conclusion