Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

Abstract

Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.

Figure 1.

Polη promotion of PCNA ubiquitination depends on PIP3 and PIP2, but not on PIP1. (A) Schematic structure of human Polη. Parts of PIP and UBZ sequences are shown. Amino-acid residues indicated by asterisks were replaced with alanines in the mutants. (B) Western blot analysis of FLAG-Polη-expressing cells. XP-V cells were transfected with the indicated plasmids for expression of FLAG-Polη (wt) or the indicated pip mutants, incubated for 24 h, irradiated with UV (15 J/m²) and further incubated for 3 h. Whole-cell lysates (WCL) or chromatin fractions were subjected to western blotting with anti-PCNA, anti-Polη and anti-Lamin B (loading control) antibodies.

Figure 2.

In vitro reconstitution of Polη–dependent PCNA ubiquitination. (A) Mono-ubiquitination reactions of PCNA were reconstituted with the indicated factors. Reaction products were analysed by western blotting with an anti-PCNA antibody. KR indicates the PCNA^K164R mutant. (B) Titration of Polη and its pip mutants. Indicated mutants were subjected to the ubiquitination assays as shown in (A). (C)Relative amounts of ubiquitinated PCNA were measured from gel images of more than three independent experiments, and the average values are plotted in the graph. Error bars show SD.

Figure 3.

DNA polymerase assays of Polη in a reconstituted system in vitro. (A) DNA replication reactions using singly primed M13 mp18 ssDNA were reconstituted with the indicated factors. The reaction products were resolved in 10% polyacrylamide gels containing 7 M urea, and visualized using a PhosphorImager. (B) Analysis of pip mutants of Polη. Indicated mutants were subjected to replication assays shown in (A) in the presence or absence of PCNA.

Figure 4.

Analysis of Polκ, Polι and their pip mutants in vitro. (A, D) Schematic structures of human Polκ (A) and Polι (D), as shown in Figure 1A. (B, E) PCNA ubiquitination assays of His-Polκ (B) and Polι (E), as shown in Figure 2. (C, F) DNA polymerase assays of His-Polκ (C) and Polι (F), as shown in Figure 3.

Figure 5.

Promotion of mono-ubiquitination of PCNA in cells by Polκ but not Polι. (A, B) Western blot analysis of Polκ-expressing cells. XP-V (A) and normal cells (B) were transfected with a plasmid to express GFP-Polκ or GFP-Polη (as a control). (C, D) Western blot analysis of Polι-expressing cells. XP-V (C) and normal cells (D) were transfected with a plasmid to express FLAG-Polι or FLAG-Polη (as a control). The transfected cells were incubated for 24 h, irradiated with UV (15 J/m²) and further incubated for 3 h. Whole-cell lysates (WCL) were subjected to western blotting with anti-PCNA, anti-Lamin B (loading control) and anti-GFP or anti-FLAG antibodies.

Figure 6.

Interactions between Y-family DNA polymerases and mUb-PCNA in vitro. (A–J) Analysis of DNA synthesis by Polη (A–F), Polι (G–H) and His-Polκ (I–J), as shown in Figure 3, in the absence or presence of PCNA (designated as PCNA or +) or mUb-PCNA (designated as uPCNA or u). Concentrations of polymerases increase in the order 0.25, 0.5, and 1 nM (A–F and I) or 1, 2, and 4 nM (G–H), or remain constant at 1 nM (J).

Figure 7.

Cellular functions of the motifs of Polη. (A) Co-localization of Polη with PCNA. XP-V cells were transiently transfected with plasmids encoding wild-type FLAG-Polη or the indicated mutants. After UV irradiation, FLAG-Polη and PCNA were visualized by immunostaining with anti-Polη and anti-PCNA antibodies, respectively. Nuclei were stained by Hoechst 33342. Scale bars represent 5 μm. Control experiments confirming expressions of FLAG-Polη were shown in Supplementary Figure S7. (B) UV sensitivities of XP-V cells stably expressing FLAG-Polη. Cells were irradiated with the indicated dose of UVC, incubated with 1 mM caffeine for 4 days, and their viabilities were measured. Error bars show SD from three independent experiments. (C) A model for a regulatory network for foci formation and the TLS function of Polη/ι/κ. Interactions of Polη/κ with PCNA, together with RAD6-(RAD18)₂, leads to their accumulation by promoting mono-ubiquitination of PCNA around stalled 3′-OH ends. Interactions of Polη/ι/κ with mUb-PCNA via PIPs and UBDs stimulate DNA synthesis at stalled 3′-OH ends. See text for details.