Résumé
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 Nterminal
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.
Spanish Ministerio de Economía
y Competitividad and the
Fondo Europeo de Desarrollo Regional (MINECO/FEDER)
[CTQ2017-83810-R to F.J.B.]; Labex EpiGenMed, an
“Investissements d”avenir’ program [ANR-10-LABX-12-01 to
PB]. MOSTMicro [LISBOA-01-0145-FEDER-007660 to
T.N.C. and H.M.]. A.G.M. acknowledges Spanish MINECO
for predoctoral contract BE-2015-075847, and the CIC
bioGUNE acknowledges MINECO for the Severo Ochoa
accreditation Sev-2016-0644. The CBS-Montpellier is a
member of France-BioImaging (FBI) and the French Infrastructure
for Integrated Structural Biology (FRISBI), two
national infrastructures supported by the French National
Research Agency (ANR-10-INSB-04-01 and ANR-10-INSB-
05, respectively).