Phosphorylation of the C subunit (p66) of human DNA polymerase d Laure Lemmens a , Serge Urbach b , Renaud Prudent c , Claude Cochet c , Giuseppe Baldacci a, * , Patrick Hughes a a Institut Curie-CNRS, Unite ´ de Genotoxicologie et Cycle Cellulaire, Universite ´ Paris Sud-XI, Batiment 110, 91405 Orsay Cedex, France b Institut de Ge ´nomique Fonctionnelle, CNRS UMR 5203, INSERM U661, Universite ´ Montpellier 1, Universite ´ Montpellier 2 141, rue de la Cardonille 34094 Montpellier Cedex 05, France c INSERM U873, CEA, iRTSV/LTS, Grenoble, F-38054, France Received 9 December 2007 Available online 26 December 2007 Abstract Of the four subunits constituting DNA polymerase d, subunit C or p66 has been shown to mainly mediate polymerase interaction with PCNA, an auxiliary factor that greatly enhances DNA polymerase d processivity on primed DNA templates. Here, we provide evidence that a highly conserved region located between amino acids 384 and 399 in the C-terminus of p66 is phosphorylated, most probably by Protein kinase CK2, and that another region, most probably located within the PCNA interacting domain in its extreme C-terminus, regulates its interaction with PCNA. Phosphorylation of p66 is associated with its co-localization with large subunit of DNA polymerase d, p125, and PCNA, to the insoluble chromatin fraction at the beginning of S-phase. Taken together, the results provide evidence that concurrent phosphorylation events in p66 may positively and negatively regulate its activity and interactions with other components of the replisome during the cell cycle. Ó 2007 Elsevier Inc. All rights reserved. Keywords: DNA polymerase d; Phosphorylation; PCNA; p66; Cell cycle regulation DNA polymerases are critical for maintaining genome stability through the processes of DNA replication and DNA repair. In eucaryotes, the bulk of genomic DNA is replicated by DNA polymerases a, d, and e [1]. Of these, DNA polymerases d and e require Proliferating Cell Nuclear Antigen (PCNA) for full processivity [2]. In the case of DNA polymerase d, a domain located in the C-ter- minus of its C subunit, termed p66, has been shown to mediate stimulation of DNA polymerase d activity by PCNA [3]. It is becoming increasingly clear that many of the pro- teins engaged in DNA synthesis undergo post-translational modifications that profoundly modify their activities. In the case of DNA polymerases, the p180 and p68 subunits of DNA polymerase a are phosphorylated by cyclin-depen- dent kinases in a cell cycle-dependent manner [4]; and DNA polymerase k, implicated in various pathways of DNA repair, is phosphorylated by the Cdk2/cyclin A com- plex [5]. Curiously however, there have been very few reports about post-translational modifications of DNA polymerase d. Previous work in our laboratory showed that the p66 subunit of DNA polymerase d could be phosphor- ylated by Cdk/cyclin complexes in vitro and that this phos- phorylation could be blocked by PCNA [3]. Here, we show that phosphorylation of p66 may also affect replisome assembly at the start of S-phase in HeLa cells. Materials and methods Reagents, peptides, buffers, and enzymes. Phosphatases were from New England Biolabs Inc. Dithiothreitol (DTT) was from Promega. Isopropyl- b-D-thiogalactopyranoside (IPTG) was from Q-Biogen. Where indicated, protease inhibitors: anti-pain, leupeptin, aprotinin and pepstatin (ICN Biomedicals Inc.) were added at 5 lg/ml. Hybridoma supernatants were 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.083 * Corresponding author. Fax: +331 69863058. E-mail address: baldacci@curie.u-psud.fr (G. Baldacci). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 367 (2008) 264–270