dna repair 6 ( 2 0 0 7 ) 1726–1731 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/dnarepair Requirements for PCNA monoubiquitination in human cell-free extracts Val ´ erie Schmutz a , J´ erˆ ome Wagner a , R´ egine Janel-Bintz a , Robert P.P. Fuchs b , Agn` es M. Cordonnier a,∗ a Institut Gilbert Laustriat, Biomol ´ ecules, Biotechnologie, Innovations th´ erapeutiques,UMR 7175 - LC1 - CNRS/ULP, ESBS, Bld S´ ebastien Brandt, BP 10413, Illkirch 67412, France b Genome Instability and Carcinogenesis, CNRS FRE 2931,Chemin Joseph Aiguier, 13402 Marseille cedex 20, France article info Article history: Received 27 April 2007 Received in revised form 11 June 2007 Accepted 13 June 2007 Published on line 31 July 2007 Keywords: PCNA Monoubiquitination Human cell-free extract Rad6/Rad18 abstract The Rad6/Rad18-dependent monoubiquitination of PCNA plays a crucial role in regulating replication past DNA damage in eukaryotic cells. We show here that in human cell-free extracts, efficient PCNA monoubiquitination requires both the synthesis of relatively long DNA tracts and polymerase idling or stalling at sites of DNA modification or DNA secondary structures. This dual dependency suggests a dynamic process in which, following initia- tion, the DNA synthesizing complex undergoes modifications that make it competent as a mediator for the activation of the Rad6/Rad18 pathway. © 2007 Elsevier B.V. All rights reserved. 1. Introduction Proliferating cell nuclear antigen (PCNA) was identified as an essential factor for both the processivity of the replicative DNA polymerase complex, and the correct coordination of the lead- ing and lagging strand synthesis [1]. PCNA is a homotrimeric ring-shaped protein complex that is loaded onto a primed template DNA containing a 3 ′ OH recessed terminus by the replication factor C, RFC [2]. Once loaded, PCNA encircles double-stranded DNA and acts as a sliding clamp that tethers its binding partners to DNA [3]. PCNA is known to interact with numerous proteins involved in different DNA transactions [4]. How these multiple partners bind PCNA in a coordinate man- ner to perform their functions in DNA replication and repair, remains an open question. ∗ Corresponding author. Tel.: +33 3 90 24 46 93; fax: +33 3 90 24 46 86. E-mail address: cordonnier@esbs.u-strasbg.fr (A.M. Cordonnier). In several organisms, PCNA has been shown to be monoubiquitinated by Rad6/Rad18 complex in response to various DNA damaging agents that block the replication forks [5–9]. Genetic epistasis analyses conducted in S. cerevisiae show that monoubiquitinated PCNA (Ub-PCNA) is involved in the same pathway as the translesional DNA polymerases pol and pol [5,10,11]. In addition, the interaction between Ub-PCNA and translesional DNA polymerases promotes lesion bypass in mammalian cells [12,13,14]. Altogether these observations point to a role of PCNA monoubiquitination in the regulation of the switch between replicative and specialized DNA poly- merases during translesion synthesis. However, the molecular events that trigger PCNA monoubiquitination as well as the exact role of Ub-PCNA in the TLS process remain elusive. The current consensus suggests that the single-stranded binding 1568-7864/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2007.06.003