Complex formation of HLTF with Rad6–Rad18 and Mms2–Ubc13. (A) Immunoprecipitation of human HLTF in complex with PCNA, Ubc13, Mms2, or Rad18. HEK293FT cells were transiently transfected with plasmids expressing HA–PCNA, HA–Ubc13, HA–Mms2, or HA–Rad18 alone or together with FLAG-HLTF as indicated. Thirty-six hours after transfection, half of the cells were treated with 40 J/m² UV, and 6 h later total cellular lysates were prepared. Immunoprecipitations (IP) were carried out with anti-FLAG antibodies, and subsequently the precipitates were analyzed by Western blots (WB) using anti-FLAG and anti-HA antibodies as indicated (lanes 1–16). (B) Purity of the proteins. One microgram of each protein was analyzed on 12% denaturing SDS-polyacrylamide gel. Lane 1, molecular mass standards; lane 2, GST–HLTF; lane 3, Rad6–Rad18 complex; lane 4, Mms2–Ubc13 complex. (C) GST pull-down of purified HLTF with Rad6–Rad18 and Mms2–Ubc13. GST–HLTF (10 μg) immobilized on glutathione-Sepharose beads was incubated with Rad6–Rad18 (7.5 μg) and Mms2–Ubc13 (6 μg). After washing, bound proteins were eluted by glutathione. Aliquots of each sample, taken before addition to the beads (L), from the flow-through fraction (F), from the last wash (W), and from the eluted proteins (E), were analyzed on a 12% SDS-polyacrylamide gel (lanes 2–5). The results for the control experiment using GST instead of GST–HLTF are shown in lanes 6–9.

HLTF is a ubiquitin ligase for Mms2–Ubc13-dependent K63-linked polyubiquitination of PCNA. (A) HLTF is required for PCNA polyubiquitination. PCNA (50 nM) was incubated in the absence or presence of various combinations of Rad6–Rad18 (100 nM), Mms2–Ubc13 (100 nM), and HLTF (100 nM) as indicated. Ubiquitination (Ub) of PCNA was followed by Western blot using anti-PCNA antibody. (B) Polyubiquitination occurs at the K164 residue of PCNA. K164R mutant PCNA (50 nM) was incubated with Rad6–Rad18 (100 nM) or with Rad6–Rad18 (100 nM), Mms2–Ubc13 (100 nM), and HLTF (100 nM) as indicated. (C) Concentration dependence of the ubiquitin ligase activity of HLTF. Polyubiquitination of PCNA (50 nM) was carried out at constant Rad6–Rad18 and Mms2–Ubc13 concentration (40 nM) but at increasing levels of HLTF (5–40 nM). (D) HLTF promotes K63-linked polyubiquitination of PCNA. PCNA ubiquitination reactions were carried out with K63A or K48A mutant ubiquitins or wild-type ubiquitin in the presence of various combinations of Rad6–Rad18, Mms2–Ubc13, and HLTF as indicated.

HLTF complements the UV sensitivity of the yeast rad5Δ mutant and contributes to UV and MMS resistance of human cells. (A) Schematic representation of HLTF, SHPRH, and Rad5 proteins. All three proteins exhibit the conserved SNF2-type helicase domain shown by gray boxes numbered I–VI and a C3HC4-type RING-finger domain represented by a black box. The HIRAN motif of HLTF and Rad5 is shown by a striped box that is followed by a leucine heptad repeat motif labeled by a black line and 3L. In SHPRH, the H1,5 domain found in linker histone 1 and histone 5 families and a PHD domain are shown by dark gray boxes. (B) Complementation of the UV sensitivity of the rad5Δ yeast strain by HLTF. The indicated yeast strains, rad30Δ, rad5Δrad30Δ, and rad5Δrad30Δ in which human HLTF was expressed, were grown until midlog phase and spotted onto plates in 10× serial dilutions at equal cell concentrations. Cells were UV-irradiated with the indicated doses and incubated in the dark at 30°C. Cell growth was evaluated after 2 days. (C) Effect of siRNA inhibition of HLTF on MMS sensitivity of human cells. Normal human fibroblasts (HF) were transfected with HLTF-specific or negative control (NC) siRNAs followed by incubation in the presence of the indicated concentration of MMS for 3 h. After 2 days, cell survival was determined by MTT assay. Data shown as mean value and the SD presented as an error bar were calculated from three independent experiments. (D) Effect of siRNA inhibition of HLTF on UV survival of human cells. Normal HF cells were transfected with HLTF siRNA and irradiated with the indicated doses of UVC light, and cell survival was measured as described above. (E) Effect of HLTF knockdown on UV survival in XPA⁻ cells. HLTF or Pol η expression was inhibited in XP-A-deficient human fibroblasts by siRNA and treated with 2 J/m² UV light before cell survival was examined. The first bar represents a control sample transfected with nonspecific siRNA.

HLTF promotes PCNA polyubiquitination in human cells. (A) PCNA polyubiquitination is stimulated by HLTF. HEK293FT cells were transfected with various combinations of plasmids expressing HA–ubiquitin, GFP–HLTF, and DsRed–Rad18 and UV-irradiated at 40 J/m². (Upper) After 48 h, cells were lysed and analyzed with anti-PCNA antibody. (Lower) From the lysates PCNA was immunoprecipitated (IP) with anti-PCNA antibody and PCNA ubiquitination was confirmed with anti-polyubiquitin (FK2) antibody. Because the IgG heavy chain migrates to the same position as HA–Ub–PCNA and is recognized by the secondary antibody, the differences in the levels of monoubiquitinated PCNA in different lanes are difficult to visualize. (B) Stimulation of polyubiquitination of PCNA by HLTF. HEK293FT cells were transfected with plasmids expressing HA–PCNA, 5His-ubiquitin, and with or without MYC–HLTF, as indicated. Forty-eight hours after transfection, cells were lysed and ubiquitinated proteins were immobilized on Ni-beads. Among the bound proteins ubiquitinated forms of PCNA were visualized by Western blot (WB) using anti-HA antibody.