Separate domains of Rev1 mediate two modes of DNA damage bypass in mammalian cells

Abstract

The Y family DNA polymerase Rev1 has been proposed to play a regulatory role in the replication of damaged templates. To elucidate the mechanism by which Rev1 promotes DNA damage bypass, we have analyzed the progression of replication on UV light-damaged DNA in mouse embryonic fibroblasts that contain a defined deletion in the N-terminal BRCT domain of Rev1 or that are deficient for Rev1. We provide evidence that Rev1 plays a coordinating role in two modes of DNA damage bypass, i.e., an early and a late pathway. The cells carrying the deletion in the BRCT domain are deficient for the early pathway, reflecting a role of the BRCT domain of Rev1 in mutagenic translesion synthesis. Rev1-deficient cells display a defect in both modes of DNA damage bypass. Despite the persistent defect in the late replicational bypass of fork-blocking (6-4)pyrimidine-pyrimidone photoproducts, overall replication is not strongly affected by Rev1 deficiency. This results in almost completely replicated templates that contain gaps encompassing the photoproducts. These gaps are inducers of DNA damage signaling leading to an irreversible G(2) arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.

FIG. 1.

UV hypersensitivity of Rev1-mutant MEF lines. The clonogenic survival of unexposed cells was set at 100%. Data represent the average of three independent experiments. WT, wild type.

FIG. 2.

UV-exposed Rev1^B/B and Rev1^−/− MEFs display similar reductions in tract lengths of DNA fibers. (A) Schematic representation of replication labeling and representative replication forks during ongoing and stalled replication. (B) Representative set of DNA fibers of wild-type (Wt), Rev1^B/B, and Rev1^−/− MEFs exposed to 0, 20, or 40 J/m² UV-C. (C) Average ratio of IdU to BrdU in wild-type, Rev1^B/B, and Rev1^−/− MEFs and Rev1 mutant MEFs complemented with mouse Rev1. (D) Distribution of percentages of replication forks at corresponding IdU/BrdU ratios. Blue bars, 0 J/m² UV-C; red bars, 20 J/m² UV-C; green bars, 40 J/m² UV-C. (E) Length of replicating forks during BrdU labeling in unexposed wild-type cells and wild-type, Rev1^B/B, and Rev1^−/− MEFs at 20, 40, and 60 min after exposure to 20 J/m² UV-C.

FIG. 3.

Delayed progression of replicons in Rev1 mutant cells. (A) Scheme of the alkaline DNA unwinding assay. Nascent DNA is pulse labeled with [³H]thymidine (dotted line) just before induction of UV damage (open and closed signs) (top). Cells were cultured in medium without label (middle). Stalling of a fork at a UV lesion results in a DNA end containing [³H]thymidine that is locally denatured using alkaline, followed by analysis using hydroxyl apatite (bottom). (B) Level of [³H]thymidine in ssDNA after alkaline unwinding. Results for mock-treated cells are shown in the left panel. Right panel shows results for cells exposed to 10 J/m² UV-C. (C) Scheme of alkaline sucrose gradient sedimentation using T4 endonuclease V. Template DNA was uniformly labeled with [¹⁴C]thymidine (solid line) followed by exposure to 10 J/m² UV-C inducing CPD (open symbols) and (6-4)PP (closed symbols) (top). Elongating daughter strands were labeled with [³H]-thymidine (dotted line; middle). At different time points, cells were lysed, and [¹⁴C]thymidine containing DNA was cleaved by T4 endonuclease V at a CPD, followed by size fractionation using alkaline sucrose gradients (bottom). The [¹⁴C]thymidine-labeled inter-CPD size distribution serves as an internal standard since CPDs are not removed in mouse cells. (D) Alkaline sucrose gradient profiles of wild-type, Rev1^B/B, and Rev1^−/− MEF lines at 15, 120, and 240 min after exposure to 10 J/m² UV-C. WT, wild type; Mw, molecular weight.

FIG. 4.

Reduced S and G₂/M phase progression in Rev1-deficient MEFs and induction of DNA damage signaling. (A) Cell cycle profiles of primary MEFs pulse labeled with BrdU immediately after exposure to 2 J/m² of UV-C, followed by chase, to determine S-phase progression. (B) Quantification of UV-C exposed cells in G₁, S, and G₂/M. (C) Western blots showing induction of Chk1 phosphorylation by UV in mutant cell lines (arrows). The asterisk indicates a cross-reacting band. β-Actin served as a loading control. WT, wild type.

FIG. 5.

Identification of strand discontinuities in Rev1-deficient MEFs. (A) At left are alkaline sucrose gradient profiles of Xpc^−/− and Rev1^−/− Xpc^−/− MEF lines at 2 h (triangles), 4 h (circles), and 6 h (squares) after exposure to 5 J/m² UV-C. Open symbols, [¹⁴C]thymidine-labeled DNA; closed symbols, [³H]thymidine-labeled DNA. Panels at right show a continuation of replication during time depicted as the ratio of [³H]thymidine counts per fraction and the total amount of [¹⁴C]thymidine counts in the gradient. (B) Alkaline DNA unwinding of DNA that was pulse labeled by [³H]thymidine before and continuously labeled for up to 24 h after exposure to 10 J/m² UV-C. For each time point, the percentage of ssDNA from total DNA of mock-treated cells was set at 100%. (C) Immunostaining of (6-4)PP and CPD in Xpc^−/− and Rev1^−/− Xpc^−/− MEFs exposed to 5 J/m² UV-C using antibodies specifically recognizing UV lesions in ssDNA. α, anti.