PCNA is monoubiquitinated in response to DNA harm and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway translesion synthesis (TLS). [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured CI-1011 cells. Moreover monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 interaction. His-tagged PCNA (His-PCNA) SAPKK3 and His-tagged BPLF1 (His-BPLF1) constructs (54) were grown in BL21 Codon Plus DE3-RIPL cells (Stratagene) and were induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 to 4 h. The cultures were centrifuged and the pellet was resuspended in HLB buffer (50 mM Na2HPO4 [pH 8.0] 20 mM imidazole 10 glycerol 0.02% NP-40 5 mM β-mercaptoethanol [BME] 150 mM CI-1011 NaCl). The cells were lysed by CI-1011 sonication and cleared lysates were incubated with nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) overnight. The beads were eluted with LB plus 300 mM imidazole and dialyzed against S100 buffer (50 mM HEPES [pH 7.6] 150 mM NaCl 10 glycerol 0.02% NP-40 5 mM BME plus protease inhibitors). Purified His-BPLF1 1-246 and His-PCNA were incubated together for 30 min at 25°C. Cell viability assay. H1299 cells were transfected with BPLF1 1-246 BPLF1 with the active site cysteine mutated (BPLF1 C61S) and BPLF1 PIP. Forty-eight hours posttransfection cells were UV irradiated with 0 1 2 5 and 10 J/m2 and were grown for 12 days. The cells were washed with PBS and counted. RESULTS PCNA interacts with BPLF1 either directly or through a complex involving both PCNA and BPLF1. It is noteworthy that the enzymatically active form of BPLF1 interacts more strongly with PCNA which suggests that functional BPLF1 is required for its interaction with CI-1011 PCNA. Fig 1 BPLF1 interacts with PCNA both and interaction immunoprecipitations (IPs) were performed on lysates from 293T cells expressing BPLF1 1-246 followed by Western blotting (WB) for endogenous levels of PCNA. Western blotting … In addition immunoprecipitations (IPs) with hemagglutinin (HA)-tagged PCNA (pCMV-HA-PCNAwt [wt stands for wild type]) (57) and FLAG-tagged BPLF1 1-246 revealed that PCNA and BPLF1 1-246 interact within the cell (Fig. 1B lanes 1 to 4). Lysates from reaction mixtures overexpressing BPLF1 and PCNA from separate samples that were mixed prior to IP demonstrate that interactions were not artifacts that occurred after cell lysis under IP conditions (Fig. 1B lane 5). PCNA interacts with BPLF1 reactions were performed. BPLF1 1-246 and PCNA were purified from and incubated together for 30 min at 25°C. Immunoprecipitations were performed with BPLF1 antibodies and probed for the presence of PCNA (Fig. 1C). The results indicate that BPLF1 1-246 interacts directly with PCNA. Deubiquitinating activity of BPLF1 removes ubiquitin from PCNA and from PCNA. The reduced levels of CI-1011 ubiquitinated PCNA observed in BPLF1-expressing cells (Fig. 2A) could have resulted from reduced Rad18-mediated E3 ligase activity or through BPLF1-mediated deubiquitination of PCNA (or both mechanisms). To distinguish between these possibilities we performed PCNA deubiquitination assays. A chromatin fraction made up of ubiquitinated PCNA from HU-treated and Rad18-overexpressing CI-1011 H1299 cells was evenly divided and incubated with or without active recombinant bacterial BPLF1 1-246 (purified from as described previously ). As shown in Fig. 2B chromatin-associated PCNA was deubiquitinated by purified BPLF1 1-246. The levels of unmodified PCNA in chromatin were unaffected by incubation with BPLF1 1-246. The results of these experiments confirm that BPLF1 has PCNA-directed DUB activity. BPLF1 DUB activity results in reduced recruitment of DNA polymerase η. Monoubiquitinated PCNA interacts much more strongly with Polη than unmodified PCNA (32). Monoubiquitinated PCNA is necessary for the DNA damage-inducible recruitment of Polη to sites of replication stalling (6 32 Therefore we predicted that deubiquitination of PCNA by BPLF1 DUB would compromise damage-induced recruitment of Polη to chromatin. To test this prediction 293 cells were transfected with a Rad18 expression plasmid to.