Published online 5 May 2007 Nucleic Acids Research, 2007, Vol. 35, No. 11 3551–3560 doi:10.1093/nar/gkm243 Involvement of DNA polymerase k in the repair of a specific subset of DNA double-strand breaks in mammalian cells Jean-Pascal Capp1, Franc¸ ois Boudsocq1, Anne-Gaelle Besnard1, Bernard S. Lopez2, Christophe Cazaux1, Jean-Se´ bastien Hoffmann1 and Yvan Canitrot1,* 1Genetic instability and Cancer group, Institute of Pharmacology and Structural Biology, University Paul Sabatier, UMR CNRS 5089, 205 route de Narbonne, 31077 Toulouse cedex and 2De´ partement de Radiobiologie et Radiopathologie, 60-68 av. Ge´ ne´ ral Leclerc, UMR 217 CNRS-CEA, 92265 Fontenay aux Roses cedex, France Received February 27, 2007; Revised April 2, 2007; Accepted April 3, 2007 ABSTRACT the recently discovered DNA polymerase l (poll) (2,3). Polm and poll share some homology and both have been The repair of DNA double-strand breaks (DSB) described as participating in vitro in the repair of DNA requires processing of the broken ends to complete double-strand breaks (DSB) by non-homologous end the ligation process. Recently, it has been shown joining (NHEJ) (4,5). The hallmark of animals or cells that DNA polymerase k (polk) and DNA polymerase j defective in one of the components of the NHEJ pathway (polj) are both involved in such processing during is an increased sensitivity to ionizing radiation (IR) (6–8). non-homologous end joining in vitro. However, no However, cells isolated from animals knocked out for poll phenotype was observed in animal models defective or polm do not present differences in sensitivity to any for either polk and/or polj. Such observations could DNA damaging agents (9–11). Furthermore, mice defec- result from a functional redundancy shared by the X tive for both poll and polm fail to reveal increased family of DNA polymerases. To avoid such redun- sensitivity to IR (10). These results suggest that an dancy and to clarify the role of polk in the end alternative pathway could deal with the DNA breaks normally managed by poll and polm, or that these joining process, we generated cells over-expressing polymerases are not involved in NHEJ in vivo. the wild type as well as an inactive form of pol k We showed, however, in a previous work that the (polkD). We observed that cell sensitivity to ionizing expression of an inactive form of poll sensitized radiation (IR) was increased when either polk or mammalian cells to IR (12). In order to better understand polkD was over-expressed. However, the genetic the cellular role of polm in NHEJ, we generated cells over- instability in response to IR increased only in cells expressing either the wild type form of polm or an inactive expressing polkD. Moreover, analysis of intrachro- form of polm to determine any interference with NHEJ. mosomal repair of the I-SceI-induced DNA DSB, did We used cells containing an integrated chromosomal not reveal any effect of either polk or polkD substrate allowing the evaluation of the I-SceI-induced expression on the efficiency of ligation of both DSB repair by NHEJ (13). We observed a sensitization of cohesive and partially complementary ends. Finally, cells expressing polmD toward IR. The joining efficiency of the sequences of the repaired ends were specifically the different DNA ends was not affected, but the affected when polk or polkD was over-expressed, molecular analysis of the repaired junctions from incom- pletely complementary DNA ends revealed a specific supporting the hypothesis that polk could be decrease in the proportion of DNA ends undergoing DNA involved in the repair of a DSB subset when synthesis. resolution of junctions requires some gap filling. MATERIALS AND METHODS INTRODUCTION Cells The DNA polymerase m (polm) (1) belongs to the X family of DNA polymerases comprising the long time identified The C’10 and A’7H cell lines were cultured in DMEM and extensively studied DNA polymerase b and medium (GIBCO BRL, Eragny, France) as previously *To whom correspondence should be addressed. Tel: þ33 5 6117 5861; Fax: þ33 5 6117 5994; Email: [email protected] Correspondence may also be addressed to Jean-Se´bastien Hoffmann. Tel: þ33 5 6117 5975; Fax: þ33 5 6117 5994; Email: [email protected] ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 3552 Nucleic Acids Research, 2007, Vol. 35, No. 11 described (13). The C’10pI2 and A’7HpI4 clones, the in PBS and fixed in PBS/2% PAF for 20 min at C’10m2 and A’7Hm2 clones and the C’10mDB and room temperature. Cells were then stained with anti- A’7HmD5 clones were obtained after transfection with H2Kd (1/30 mouse isotype, SF1-1.1, Pharmigen), or anti- the empty pIRESpuro2 vector (Clontech, Heidelberg, CD4 (1/30 rat isotype, H129.19, Pharmigen), or anti-CD8 Germany) or the pIRESpuro2 vector containing the (1/30 rat isotype, 53-6.7, Pharmigen) for 30 min at room cDNA coding for the human wild-type DNA polymerase temperature in PBS/0.5% BSA. Cells were then incubated m (polm) or the inactive form of DNA polymerase m with anti-mouse-FITC (1/530 mouse isotype, F-2761, (polmD), respectively. Individual clones were obtained Molecular probe) for 30 min at room temperature. The after transfection with jetPEI (Qbiogen, Illkirch, France) frequency of H2K events allowed us to estimate the and puromycin selection (5 mg/ml). efficiency of I-SceI transfection and activity. Scoring of the NHEJ events affecting CD4 and CD8 was performed Generation of cells expressing polk isoforms by flow cytometry using FACScan (Becton Dickinson, Polm expressing plasmids pIRES-polm and pIRES-polmD San Diego, CA, USA). were constructed by PCR amplification from a P17-His vector plasmid carrying the cDNA sequence of the polm Enrichment of CD4þ -expressing cells by MACS gene (3). The polm catalytically inactive mutants were Cells were dissociated with PBS/EDTA (50 mM) and constructed from P17-His plasmid, using the Quick washed with PBS/0.5% BSA/2 mM EDTA. Cells (1 Â 106) Change mutagenesis kit (Stratagene, La Jolla, CA, USA) were stained with anti-CD4 for 15 min at room tempera- according to the manufacturer’s instructions. Residues ture, then incubated with beads coated with goat anti-rat D330 and D332 in polm were replaced by Ala using two IgG (Miltenyi Biotec, Bergisch Gladbach, Germany) primers also containing a new BsmI site used for mutation in PBS/0.5% BSA/2 mM EDTA for 15 min at room selection. The resulting constructs were sequenced and temperature. After washing, the positively stained cells error-free vectors were used to transfect cells. were separated onto miniMACS columns and enriched by 30%. DNA polymerase purification and activity assay The WT and catalytically mutated forms of polm on p17 Junction sequence analysis plasmid were sequenced and introduced into BL21(DE3) Genomic DNA was prepared from a population of CD4 bacteria before purification. The different polm enriched cells (Puregene, Gentra Systems, Hameenlinna, proteins were purified by Nickel chelation chromatogra- Finland), and junctions of deletion events (CD4þ) phy according to Novagen’s suggestions with the were amplified by PCR with the following primers following modifications. Cells were lysed by freezing (CMV1: 50-TGGCCCGCCTGGCATTATGCCCAG-30 for 16 h at À708C and slowly thawing on ice in the and CD4int: 50-GCTGCCCCAGAATCTTCCTCT-30). presence of 20 mg/ml lysozyme and 1% Triton X100. PCR products of 800 bp were obtained after 35 cycles of Proteins were concentrated and stored in buffer containing amplification with Taq polymerase (Biolabs, Beverly, MA, 50 mM Tris pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 1 mM USA) (13). The PCR products were cloned into Topo-TA DTT and 20% Glycerol. Protein purity was estimated system (Invitrogen, Carlsbad, CA, USA), which allows as 490% by visual inspection of Coomassie Blue-stained isolation of individual clones, and sequenced on one 12% SDS–polyacrylamide gels. Standard primer strand. extension reactions were performed at 378C for 1 h as previously described (14). Cytotoxicity studies Western blotting Cytotoxicities were determined by clonogenic assay (16). For IR, cells (400 or 800) were plated in 25 cm2 flasks and Total cellular protein extracts (50 mg) were separated by allowed to attach overnight. Cells were subsequently 10% SDS-PAGE (PolyAcrylamide Gel Electrophoresis) irradiated in growth medium with a Co-source irradiator and transferred to PVDF membrane. Polm was detected (IBL 437C type H, Oris industries SA, Gif sur Yvette, using polyclonal antibody (1/500) (AbCam, Cambridge, France). For camptothecin (CPT), mitomycin C (MMC) UK) followed by incubation with horseradish peroxidase and methyl methanesulfonate (MMS) (Sigma, France) (HRP)-conjugated anti-rabbit IgG, and revealed using the cytotoxicities, cells (400) were plated in 6-well plates and ECL system (Amersham, Freiburg, Germany). Equal allowed to attach overnight. Cells were treated in growth loading was determined using monoclonal anti-actin medium with increasing concentrations of drug for 24 h antibody (1/5000) (AC10, Sigma, Lyon, France). (for CPT) or 1 h (for MMS and MMC). After 8 days post- treatment incubation, the plates were washed with PBS Measurement of NHEJ by FACS after induction of DSBs and colonies were fixed with methanol, stained with Cells (5 Â 105) were plated and allowed to attach crystal violet solution and colonies of more than 50 cells overnight. Expression of the meganuclease I-SceI in cells were counted. Survival was expressed as the plating was achieved by transient transfection of the expression efficiency of treated cells relative to the untreated control plasmid pCMV –I-SceI (15) using Jet-PEI (Q-BIOgen, cells.