PCNA[KR] cells are sensitive to DNA-damaging agents.

<p>The sensitivities of WI38VA13-derived cells to UV irradiation (A) or cisplatin (Cis-Pt) (B) with and without siRNA-mediated knockdown of endogenous PCNA (siPCNA). Clonogenic survival rates were determined after UVC irradiation or cisplatin treatment (24 h) of the indicated cell strains. Data are represented as the mean ± standard deviation of n = 3 independent experiments.</p

PCNA[KR] cells show DNA replication problems after UV irradiation.

<p>(A, B) S-phase progression analysis. Vector-transfected, PCNA-wild-type (WT) or PCNA[KR] WI38VA13 cells were synchronized using the double thymidine block method, mock-irradiated (UV-) or irradiated with UVC at the released time point, and then incubated for the indicated periods prior to FACS analysis of the cell-cycle profile. (C) Immunoblot analysis of CHK1 phosphorylation. Cells were irradiated with 10 J/m² UVC and incubated for the indicated periods. Immunoblotting was performed using anti-phospho-CHK1 (Ser345), anti-CHK1 and anti-actin antibodies. (D) Analysis of γH2AX-positive populations. The indicated cells were irradiated with 5 J/m² UVC and incubated for the indicated periods. The cells were then fixed and stained with an anti-γH2AX antibody (shown in red). Nuclei were visualized by Hoechst 33342 staining (shown in blue).</p

Ectopic K164R-mutated PCNA forms hetero-trimers with endogenous PCNA, depletes endogenous PCNA homo-trimers, and disturbs DDT.

<p>(A) Immunoprecipitation assays showing the formation of hetero-trimers of exogenous PCNA[KR] and endogenous PCNA in WI38VA13 cell transformants. The chromatin fractions (20% input) prepared from vector-transfected cells (v) and cells expressing varying levels of HA-PCNA[KR] (clone #1 = #2 > #3) were immunoprecipitated using an anti-HA antibody and then analyzed by immunoblotting using an anti-PCNA, anti-HA, or anti-Lamin B (control) antibody. The whole cell lysates (WCLs) and 20% unbound samples were also analyzed. The ratios of the signal intensities of endogenous PCNA detected in the unbound fractions of clones #1, #2, and #3 to control cells (v) are indicated below the upper panel. (B) Cell-cycle progression analyses of vector-transfected WI38VA13 cells and the HA-PCNA[KR] clone #1, #2 and #3 cells. The cells were synchronized by the double thymidine block method, mock-irradiated (UV-) or irradiated with 8 J/m² UVC (UV+) at the released time point, and then incubated for the indicated periods. The cell-cycle profiles were obtained by FACS analysis. The percentages of cells in the S-phase at the 15 h time point are indicated.</p

Forced increase of mono-ubiquitinated PCNA does not restore the DDT defects of PCNA[KR] cells.

<p>PCNA-wild-type (WT) and PCNA[KR] cells were transfected with empty vector (v) or FLAG-Polη (η) or FLAG-RAD18 (18) expression constructs. (A) Cells were mock-irradiated (UV-) or irradiated with 15 J/m² UVC (UV+), incubated for 3 h, and then subjected to immunoblotting using an anti-PCNA, anti-Polη, anti-RAD18, and anti-Lamin B antibodies. (B) Cells were co-transfected with GFP and empty vector or the FLAG-Polη or FLAG-RAD18 expression construct, and then treated with 2.5 mM thymidine for 24 h to concentrate the G1/S-phase populations. The cells were then mock-irradiated (UV-) or irradiated with 8 J/m² UV and cultured for the indicated periods. The cell-cycle profiles of the GFP-positive populations were determined by FACS analysis. The percentages of cells in the S-phase populations at the 12 h time point are indicated. (C) Cells were co-transfected with His-Ub and empty vector (v) or the FLAG-Polη (η) expression construct, mock-irradiated (-) or irradiated with 15 J/m² UVC (+), and then incubated for 3 h. Ni-pull-down assays were performed using the chromatin fractions (10% input) and samples were analyzed by immunoblotting using an anti-PCNA or anti-Polη antibody.</p

PCNA homo-trimers undergo multiple-mono-ubiquitinations <i>in vivo</i>.

<p>The results of a pull-down assay using stably-expressed His-Ub. WI38VA13 cells stably expressing His-Ub (His-Ub) or vector-transfected control (vec) were mock-irradiated (-) or irradiated with 15 J/m² UVC (+) and incubated for 3 h. Ni-pull-down assays were performed using the chromatin fractions (2% input) and samples were analyzed by immunoblotting using an anti-PCNA or anti-Ub antibody. WCL, whole cell lysate.</p

Ectopic expression of K164R-mutated PCNA disturbs multiple mono-ubiquitinations of PCNA trimers in human cells.

<p>(A) Immunoblot analyses of endogenous and ectopically expressed PCNA in vector-transfected, PCNA-wild-type (WT)-expressing and PCNA[KR]-expressing WI38VA13 cells that were untreated (untreat) or transfected with a nontargeting control (NTC) or PCNA-specific (siPCNA) siRNA. Four days after transfection, the cells were mock-irradiated (UV-) or irradiated with UVC at 10 J/m² (UV+), and then incubated for a further 3 h. Lamin B was detected as a loading control. (B) Ni-pull-down assay of PCNA-WT and PCNA[KR] cells transiently transfected with empty vector (vec) or the His-Ub expression construct (His-Ub). The cells were incubated for 36 h, mock-irradiated (-) or irradiated with 15 J/m² UVC (+), and then incubated for a further 3 h. Ni-pull-down assays were performed using the chromatin fractions (5% input) and samples were analyzed by immunoblotting using an anti-PCNA or anti-Ub antibody. WCL, whole cell lysate.</p

The DDT pathway disturbed by expression of PCNA[KR] is distinct from Polη- and Rev1-mediated TLS.

<p>(A) Immunoblot analyses of PCNA expression in Polη-deficient XP2SASV3 cells stably expressing empty vector (vec), HA-PCNA-wild-type (WT) or HA-PCNA[KR] (KR). The cells were mock-irradiated (UV-) or irradiated with UVC at 10 J/m² (UV+) and then incubated for 3 h. Whole cell lysates were analyzed by immunoblotting using an anti-PCNA or anti-Lamin B (control) antibody. (B) The UVC sensitivities of Polη-deficient XP2SASV3-derived cells (XP-V) expressing empty vector, HA-PCNA-WT or HA-PCNA[KR] after treatment with an siRNA to knockdown endogenous PCNA (siPCNA) or a nontargeting control siRNA (NTC). Data are represented as the mean ± standard deviation (SD) of n = 3 independent experiments. (C) GFP-Polη was transiently expressed in WI38VA13/PCNA-WT or WI38VA13/PCNA[KR] cells treated with an siPCNA or a NTC. The cells were irradiated with 15 J/m² UVC and then incubated for 3 h. After extraction of Triton-soluble materials and fixation, PCNA was detected using an anti-PCNA antibody and nuclei were visualized by Hoechst 33342 staining. A typical nucleus of each cell type is shown. (D) Immunoblot analyses of REV1 and Lamin B (control) expression in WI38VA13 cells expressing empty vector (vec), PCNA-WT (WT) or PCNA[KR] (KR), and transfected with or without a REV1-specific siRNA. Four days after transfection, whole cell lysates were analyzed by immunoblotting using an anti-REV1 or anti-Lamin B antibody. (E) The effects of knockdown of REV1 on the UV sensitivities of the cells described in (D). The ratios of surviving cells exposed to 8 J/m² of UVC irradiation to those that were mock-irradiated were determined. The survival rates of the cells that were not treated with the REV1-specific siRNA (untreat) were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118775#pone.0118775.g004" target="_blank">Fig. 4A</a>. Data are represented as the mean ± SD of n = 3 independent experiments. (F) Schematic illustration of PCNA mono-ubiquitination-mediated DDT pathways in human cells.</p

[Erratum] Structure and mechanism of human DNA polymerase η (Nature (2010) 465 (1044-1048))

It was brought to our attention by D. Guillaume (Chimie Thérapeutique, CNRS) that deoxyribose C4′ of the cyclobutane pyrimidine dimer (CPD) photoproduct in the TT2 structure (Protein Data Bank accession number PDB 3MR4) has an inverted configuration. After further refinement, we obtained the correct sugar configuration (see Fig. 1 below). A new accession number (PDB 3SI8) has been given to the corrected structure.</p

Simultaneous disruption of two DNA polymerases, Pol? and Pol?, in Avian DT40 cells unmasks the role of Pol? in cellular response to various DNA lesions

Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase ?, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ? by generating POL?-/-/POL?-/- cells from the chicken DT40 cell line. POL?-/- cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Pol? plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POL?-/-/POL?-/- cells shows that, unexpectedly, the loss of Pol? significantly rescued all mutant phenotypes of POL?-/- cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Pol? contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells

[Erratum] Structure and mechanism of human DNA polymerase η (Nature (2010) 465 (1044-1048))

It was brought to our attention by D. Guillaume (Chimie Thérapeutique, CNRS) that deoxyribose C4′ of the cyclobutane pyrimidine dimer (CPD) photoproduct in the TT2 structure (Protein Data Bank accession number PDB 3MR4) has an inverted configuration. After further refinement, we obtained the correct sugar configuration (see Fig. 1 below). A new accession number (PDB 3SI8) has been given to the corrected structure.</p