PCNA polyubiquitination is enhanced by DNA damage that stalls DNA replication forks. (A) Distinct patterns of PCNA ubiquitination result in activation of either TS or TLS of postreplication repair. R18, RAD18; R5, RAD5; R6, RAD6; 13, UBC13; 2, MMS2. (B) Human HEK293T cells were treated with DNA-damaging agents, including 2 mM hydroxyurea (HU), 60 J/m² of UV irradiation, 0.01% MMS, 10 μg/ml aphidicolin, or 20 Gy of γ irradiation, and the chromatin-bound form of PCNA was immunoprecipitated with a monoclonal anti-PCNA antibody (PC10). The PCNA ubiquitination was detected by HRP-conjugated anti-PCNA and anti-Ub (P4D1) antibodies. Asterisks indicate nonspecific bands. PCNA polyubiquitination was enhanced in a dose-dependent (C) and time-dependent (D) manner. Ub, Ub², and Ub^N indicate mono-, di-, and polyubiquitinated species of PCNA, respectively. (E) The BrdU-incorporated flow cytometric analysis of HEK293T cell population to monitor the progression of DNA replication. HEK293T cells were treated with various DNA-damaging agents, labeled with BrdU for 1 h, and BrdU incorporation was measured on a FACS LSRII. The x and y axes represent DNA content as measured by 7-amino-actinomycin D staining and cells undergoing DNA replication as measured by BrdU incorporation, respectively. S in the rectangles represents the S phase of the cell cycle.

Ectopic expression of human HLTF and SHPRH promotes polyubiquitination of PCNA at K164 with K63-linked polyubiquitin chains. (A) Schematic representation of human (Hs) HLTF, SHPRH, and yeast (Sc) Rad5. SWI2/SNF2 (subdomains I, Ia, II, III, IV, V, and VI) and RING domains are indicated. An alignment of the RING finger domains is shown below. Conserved cysteines and histidine (C₃HC₄) are indicated with dots. (B) HLTF promotes PCNA polyubiquitination. HEK293T cells were transfected with HA-ubiquitin (HA-Ub, 0.5 μg), FLAG-PCNA (0.5 μg), HLTF-myc-His (2.0 μg), RAD6-HA (100 ng), UBC13-HA (50 ng), UBC13(C87A)-HA (50 ng), and MMS2-HA (50 ng) in the indicated combinations. PCNA (anti-FLAG) immunoprecipitates were blotted with an anti-HA antibody. Ub and Ub^N indicate mono- and polyubiquitinated species of PCNA, respectively. Expression of transfected constructs was confirmed by blotting total lysates with respective antiepitope tag antibodies. (C) HLTF and SHPRH cooperatively promote PCNA polyubiquitination. HLTF-myc-His (1.0 μg), SHPRH-myc-His (1.0 μg), or both were expressed along with HA-Ub, UBC13, and RAD6 as indicated. (D) Specificity of ubiquitin ligase activity in HLTF. Similar to (A), HEK293T cells were transfected with a PCNA-K164R mutant (left panels), or HA-ubiquitin mutants (right panels), and ubiquitinated species of PCNA were detected with an anti-HA antibody. Endogenous PCNA was immunoprecipitated in the right panel. + and − in boxes represent presence and absence of DNA listed in the right panel in transfection. 2 and 1 in boxes indicate the fold difference of DNA amount used for transfection.

SHPRH and HLTF catalyze endogenous PCNA ubiquitination. (A) The reduction of HLTF or SHPRH expression by siRNA reduced PCNA polyubiquitination. Depletion of HLTF and SHPRH was achieved by siRNA in HEK293T cells. Seventy-two hours after transfection, cells were either mock treated or treated with 0.01% MMS. Cells were harvested 1.5 h later. Anti-PCNA immunoprecipitations were performed as described in Fig. 1. Cyb is a control siRNA transfection targeting cyclophilin B that does not have any known function in DNA metabolism. The efficiency of siRNA knockdown of SHPRH or HLTF was checked by Western blot with specific antibodies. Asterisks indicate nonspecific bands. (B) Reduced PCNA polyubiquitination in SW480 cells was enhanced when HLTF expression was restored. − and + represent the absence and presence of 5-azacytidine treatment. The restoration of HLTF expression was confirmed by both Western blots and quantitative RT-PCR (qRT-PCR). β-actin (ACTB) was used as a loading control. The number indicates fold induction, measured by qRT-PCR. (C) PCNA ubiquitination is significantly reduced in the Shprh^−/− and Hltf^−/− MEF cells. MEFs were treated with mock or 60 J/m² of UV irradiation as indicated. Ub, Ub², and Ub^N indicate mono-, di-, and polyubiquitinated species of PCNA, respectively.

Reduced expression of SHPRH or HLTF enhances genomic instability and mutagenesis but not homologous recombination (HR). (A) Ramos cells expressing low levels of HLTF or SHPRH increased mutagenesis as measured by the loss of expressing surface IgM. (B) Expression of HLTF and SHPRH were reduced by shRNA (C) HLTF- and SHPRH-depleted cells showed no significant change in the frequency of HR according to FACS analysis of GFP-positive cells relative to the red fluorescent cells (transfection control). Depletion of each gene was achieved by siRNA-mediated gene knockdown. Vec and I-SceI represent transfections of vector (no DSB) and I-SceI (DSB). HR frequency was normalized to NC (nontargeting siRNA). (D) Cells expressing low levels of HLTF by shRNA showed higher sensitivity to MMS. (E) HLTF ^−/− MEF cells showed elevated levels of abnormal chromosomes after MMS treatment. MEFs were treated with 0.01% MMS for 1 h, and the metaphase spreads were analyzed 24 h after treatment. Examples of metaphase spreads from wild-type (+/+) and homozygous null (−/−) cells are shown in the left panel. The percentage of chromosome breakages and fusions from 100 metaphases are presented as graphs in the right panel.