PCNA Ubiquitination and REV1 Define Temporally Distinct Mechanisms for Controlling Translesion Synthesis in the Avian Cell Line DT40  Charlotte E. Edmunds, Laura J. Simpson, Julian E. Sale  Molecular Cell  Volume 30, Issue 4, Pages 519-529 (May 2008) DOI: 10.1016/j.molcel.2008.03.024 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Epistatic Analysis of the Role of PCNA Ubiquitination and REV1 in DNA Damage Tolerance Sensitivity of wild-type (squares), rev1 (diamonds), pcnaK164R (circles), and rev1/pcnaK164R (triangles) cells to (A) 254 nm light (UV), (B) methylmethane sulphonate (MMS), and (C) 4-nitroquinoline-1-oxide (NQO). Each point represents the mean of three independent experiments, and the error bar represents one standard deviation. For clarity, only the positive error is shown. Molecular Cell 2008 30, 519-529DOI: (10.1016/j.molcel.2008.03.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Postreplication Repair in TLS Mutants of DT40 Wild-type (A), rad18 (B), pcnaK164R (C), polη (D) and rev1 (E), cells were irradiated with 4 Jm−2 254 nm light and pulse labeled with [3H]thymidine for 20 min and either lysed immediately (UV 0′, black circles) or chased for 90 min in medium containing 10 μM thymidine before lysis (UV 90′, red triangles). Sham-irradiated cells were also chased for 90 min to assess incorporation of the label into higher molecular weight DNA in the absence of exogenous DNA damage (no UV 90′, blue triangles). The fractions in which phage λ (48 kb) and T4 genomic DNA (166 kb) show peak sedimentation are indicated by black bars. Molecular Cell 2008 30, 519-529DOI: (10.1016/j.molcel.2008.03.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Analysis of DNA Replication Following Damage by DNA Fiber Labeling in DT40 Cells (A) Schematic of the DNA fiber labeling experiment. The upper panel represents the two labeling periods and unimpeded replication with the ratio of CldU-labeled tract to IdU-labeled tract being 1:1. The lower panel illustrates a replication stall during the second labeling period showing a ratio of ∼3:1. Representative images of actual fibers are shown on the right. The length of the CldU tract in both cases is ∼20 kb. CldU, chlorodeoxyuridine; IdU, iododeoxyuridine; NQO, 4-nitroquinoline-1-oxide. (B and C) Representative field of labeled DNA fibers from DT40 cells without (B) and with (C) 25 μg/ml NQO in the second labeling period. The scale bar is 20 μM, representing ∼52 kbp (Jackson and Pombo, 1998). (D) Replication speeds in the TLS mutants used in this study. These were calculated assuming the fiber stretching determined by Jackson and Pombo for the fiber combing method employed in this article from the CldU tracts of at least 75 fibers. The error bars represent one standard deviation. (E and F) Dose response of replication arrest in wild-type DT40 at three doses of 254 nm UV light (E) and NQO (F). Molecular Cell 2008 30, 519-529DOI: (10.1016/j.molcel.2008.03.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Replication Fork Progression on Damaged DNA in Cells Deficient in PCNA Ubiquitination and REV1 (A and B) Replication stalling in response to (A) 20 Jm−2 254 nm light (red bars) or sham irradiation (blue bars) and (B) 5 μg/ml NQO (red bars) or acetone (blue bars) in DT40 TLS mutants. Each data set is derived from measurement of at least 75 fibers. (C and D) The data for 20 Jm−2 UV light (C) and 5 μg/ml NQO (D) replotted as a cumulative percentage of forks at each ratio. The p value of the two-sample Kolmogorov-Smirnov (K-S) test for the ratio distribution of each mutant, for both UV and NQO, compared to wild-type is shown adjacent to the key. Molecular Cell 2008 30, 519-529DOI: (10.1016/j.molcel.2008.03.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Domain Analysis of REV1 (A) Domain arrangement of human REV1. The sequence of the human and chicken REV1 ubiquitin-binding motifs is shown, and the residues mutated (L946A/P947A and L1024A/P1025A) to inactivate ubiquitin binding (Guo et al., 2006b) are indicated. (B) Replication stalling in rev1 mutants reconstituted with the indicated hREV1 constructs (see legend to Figure 3). (C) Data from Figure 4B for 5 μg/ml NQO presented as a cumulative percentage of forks at each ratio. The wild-type and rev1 curves from Figure 3B are shown for comparison. Molecular Cell 2008 30, 519-529DOI: (10.1016/j.molcel.2008.03.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Contribution of the Polymerase- and Ubiquitin-Binding Domains of REV1 to Replication Fork Progression on Damaged DNA (A) Dissection of the C terminus of REV1 with respect to replication stalling and survival (see also legend to Figure 3). (B) Data from Figure 4B for 5 μg/ml NQO presented as a cumulative percentage of forks at each ratio. The wild-type and rev1 curves from Figure 3B are shown for comparison. (C) Sensitivity of WT (squares), rev1 (diamonds), and rev1 complemented with YFP-hREV1 amino acids 1–1041 (circles) and with YFP-hREV1 amino acids 1–1137 (triangles) to NQO. (D) Sensitivity of WT and rev1 cells compared to rev1 complemented with YFP-hREV1(L946A/P947A/L1024A/P1025A) (hREV1ΔUBM) (triangles). A second hREV1ΔUBM clone behaved identically (data not shown). The experiments in (D) were carried out with those shown in Figure 1C, and the wild-type and rev1 curves are reproduced here for comparison. The error bars represent one standard deviation. (E) Replication stalling in polη mutants. p values for the comparison to wild-type using the K-S test are indicated. Molecular Cell 2008 30, 519-529DOI: (10.1016/j.molcel.2008.03.024) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Simplified Model for the Control of TLS at Stalled Replication Forks and Postreplicative Gaps (A) An encounter between the replicative polymerases and a stalling DNA lesion results in the recruitment of REV1 or the alteration of a preexisting complex containing REV1, possibly in response the ubiquitination or phosphorylation of proteins adjacent to the replisome (Hirano and Sugimoto, 2006; Sabbioneda et al., 2007). The C terminus of REV1 provides a stable platform for TLS by a specialized polymerase, illustrated here by Polη. Once the lesion has been bypassed, a process that may require additional polymerase switches, replicative DNA synthesis is resumed. (B) Postreplicative gaps may be detected by the single-strand DNA-binding activity of RAD18 (Bailly et al., 1997). Along with RAD6, RAD18 ubiquitinates PCNA and simultaneously delivers Polη to the primer terminus (Watanabe et al., 2004). A robust interaction between Polη and ubiquitinated PCNA allows gap filling to take place efficiently. Molecular Cell 2008 30, 519-529DOI: (10.1016/j.molcel.2008.03.024) Copyright © 2008 Elsevier Inc. Terms and Conditions