DNA Repair 8 (2009) 190–201 Contents lists available at ScienceDirect DNA Repair journal homepage: www.elsevier.com/locate/dnarepair Dynamic localization of human RAD18 during the cell cycle and a functional connection with DNA double-strand break repair Akiko Inagaki a , Wiggert A. van Cappellen a , Roald van der Laan b , Adriaan B. Houtsmuller c , Jan H.J. Hoeijmakers b , J. Anton Grootegoed a , Willy M. Baarends a,∗ a Department of Reproduction and Development, Erasmus MC, University Medical Center, PO Box 2040, 3000 CA, Rotterdam, The Netherlands b Department of Cell Biology and Genetics, Erasmus MC, University Medical Center, Rotterdam, The Netherlands c Department of Pathology, Josephine Nefkens Institute Erasmus MC, University Medical Center, Rotterdam, The Netherlands article info Article history: Received 29 July 2008 Received in revised form 16 September 2008 Accepted 6 October 2008 Available online 2 December 2008 Keywords: Cell cycle DNA double-strand breaks PCNA RAD18 abstract The ubiquitin ligase RAD18 is involved in different DNA repair processes. Here, we show that in G1 phase, human RAD18 accumulates in a few relatively large spontaneous foci that contain proteins involved in double-strand break (DSB) repair. These foci persist until cells enter S phase, when numerous small foci appear. At these sites, only 20% of RAD18 colocalizes with PCNA, a known RAD18 substrate. In late G2 phase, RAD18 relocates to nucleoli. After UVC irradiation, PCNA accumulates at the damaged site, followed by RAD18, independent of the cell cycle phase. After induction of DSBs, using low-power multi-photon laser, RAD18 accumulated at the DSB sites, but no PCNA accumulation was observed. Our data show that RAD18 accumulates on DSBs independent of the cell cycle phase. DSBs marked by RAD18 and RAD51 are also positive for RPA in G1 phase, and these DSBs persist until S phase. In addition, we show that DSBs generated in G2 phase are not all repaired, and are observed again in the next G1 phase. We conclude that repair of induced and spontaneous DSBs that accumulate RAD18 and RAD51 in G1 phase cells is delayed until S phase. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Cells are constantly exposed to internal and external factors that may cause DNA damage. Various DNA repair pathways are essential for progress through the cell cycle and long-term survival. During DNA replication, the presence of unrepaired DNA lesions threat- ens to block progression of the replication machinery. Replicative damage bypass (RDB) is a special pathway that allows progres- sion of DNA replication in the presence of DNA damage (reviewed in [1]). In Saccharomyces cerevisiae, the E2 ubiquitin-conjugating enzyme Rad6 is essential for this pathway. Depending on inter- actions with downstream components, error-free or error-prone subpathways can be activated. The first step in both pathways involves mono-ubiquitylation of PCNA by the Rad6-Rad18 complex, in which Rad18 acts as an ubiquitin ligase (E3 enzyme) [2]. PCNA forms a homo-trimer that encircles double-stranded DNA, and operates as a sliding clamp to keep the DNA polymerase machinery firmly on the DNA during DNA replication (reviewed in [3]). Mono- ubiquitylation of PCNA by the Rad6-Rad18 complex recruits specific ∗ Corresponding author. Tel.: +31 107043976; fax: +31 107044736. E-mail address: w.baarends@erasmusmc.nl (W.M. Baarends). translesion synthesis polymerases that can incorporate nucleotides in the strand opposite the site of the DNA lesions. Depending on which polymerase is recruited, this process may be error-prone or error-free (reviewed in [4,5]). Alternatively, mono-ubiquitylation by the Rad6-Rad18 complex may be followed by Rad5-Mms2- Ubc13-mediated poly-ubiquitylation [2]. The ubiquitin ligase Rad5 interacts with both the Rad6-Rad18 and the Mms2-Ubc13 com- plexes to stimulate poly-ubiquitination of PCNA. Subsequently, Rad5 is involved in error-free bypass of the damage. In mammalian cells, RAD18 complexes with the Rad6 homologs HR6A (UBE2A) and HR6B (UBE2B) [6], and regulates PCNA mono- ubiquitylation [7,8]. RAD18-knockout cells are sensitive to UVC light exposure [9,10], camptothecin, and ionizing radiation (IR) [11], that induce distortions of DNA geometry, single-strand breaks (SSBs), and double-strand breaks (DSBs), respectively. Thus, in mammalian cells, RAD18 is required for survival after the induction of almost any type of DNA damage, and this appears to be associated with novel RAD18 functions outside the context of RDB. However, the nature of these functions is still unclear. Nakajima et al. [12] showed that RAD18 accumulates at sites of SSBs, in an S-phase- independent manner. Shiomi et al. [11] also provided evidence for RAD18 functions at sites of SSBs, but restricted to S phase and PCNA independent. In addition, at DSB repair sites during S phase, 1568-7864/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2008.10.008