Accumulation of RFC complex in chromatin fraction and modification of RFC2 following treatment of 293A cells with DNA-damaging agents. A, 293A cells transfected with a FLAG epitope-tagged form of each subunit of RFC and RLCs were irradiated with UV light (lanes 3 and 9) or γ-ray (lanes 6 and 12) or treated with Me₂SO (DMSO; lanes 1, 4, 7, and 10), MMS (lanes 2 and 8), or HU (lanes 5 and 11) for 8 h. Cell extracts recovered from transfected cells were then separated into chromatin (lanes 7–12) and soluble (Sup; lanes 1–6) fractions and analyzed by Western blotting with anti-FLAG. Cell extracts recovered from RFC4-transfected cells were also analyzed by Western blotting with anti-tubulin or anti-histone H3 (lowest two blots). B, 293A cells were irradiated with UV light (lane 3) or γ-ray (lane 5) or treated with Me₂SO (lane 1), MMS (lane 2), or HU (lane 4) for 8 h. Cell extracts recovered from transfected cells were then separated into chromatin and soluble (Sup) fractions and analyzed by Western blotting either with anti-RFC1, anti-RAD17, or anti-RFC2. The arrowheads indicate the position of molecular mass markers (kDa). C, 293A cells transfected with pCDNA3·RFC2-HA were treated with the indicated dose of MMS for 8 h. Chromatin fractions from the resulting cells were analyzed by immunoblotting with anti-RFC2 or anti-PCNA. The arrowheads indicate the position of molecular mass markers (kDa). D, 293A cells transfected with pCDNA3·RFC2-HA were treated with 0.85 mm MMS for 1 h (lanes 2–5) or UV light-irradiated at 254 nm with 30 J m^-2 (lanes 6–9) and then incubated for the indicated times. Chromatin fractions were prepared and analyzed by Western blotting with anti-RFC2 and anti-PCNA. Cells treated with Me₂SO (lane 1) are shown as control. The arrowheads indicate the position of molecular mass markers (kDa). E, 293A cells transfected with pCDNA3·RFC2-HA were treated with various genotoxic agents. Chromatin fractions were prepared and analyzed by Western blotting with anti-RFC2 or anti-PCNA. The arrowheads indicate the position of molecular mass markers (kDa). Gy, grays; MMC, mitomycin C; EMS, ethyl methanesulfonate; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; Bleo, bleomycin; CPT, camptothecin.

DNA damage-independent monoubiquitylation of hRFC2 D228A. A, schematic diagram and tertiary model of human (Hs) RFC2 showing the location of Asp-228 and the sequences of the surrounding regions. Corresponding sequences for S. cerevisiae (Sc) RFC2(p40) and mouse (Mm) RFC2 homologues are also shown. The conserved Sensor 2 helix is represented by a box, and the location of the conserved SRC motif is indicated by an arrow. Asp-228 of hRFC2, shown in red, corresponds to S. cerevisiae Asp-201, which shows synthetic lethality with mutation in Rpa-1(rfa1-Y29H). There are seven conserved RFC boxes numbered consecutively from the N terminus to C terminus. B, 293A cells were transfected with expression vectors encoding wild-type (lanes 2 and 6), D228N (lanes 3 and 7), or D228A (lanes 4 and 8) forms of hRFC2-HA. 24 h after transfection cells were harvested and separated into chromatin (lanes 5–8) and soluble fractions (lanes 1–4) and then immunoblotted with anti-RFC2 or anti-PCNA antibody. The arrowheads indicate the position of molecular mass markers (kDa). C, Western blot of lysates from HCT116 cells (WILD) or RAD18-deficient HCT116 cells (RAD18^-/-). HCT116 cells transfected with pCAGGS·hRFC2(Asp-228) were treated with 0.85 mm MMS for 8 h. Chromatin fractions from the resulting cells were analyzed by Western blotting with anti-RFC2 antibody. The arrowheads indicate the position of molecular mass markers (kDa). DMSO, Me₂SO.

A model for human clamp loader-clamp complex. Ribbon (RFC2) and wire (Cα trace, RFC1, RFC3–5, and PCNA) representations of the homology model for human RFC1–5-PCNA complex are shown. The five subunits of each clamp loader complex are denoted. The colors for each of the subunits are as follows with the helical collar domains (gray) at the top of the figure: pink, RFC1; navy, RFC2; red, RFC3; green, RFC4; orange, RFC5; gold, PCNA. The side chain atoms of Asp-228 of RFC2 are indicated as balls in cyan. A, a side view of the clamp loader-clamp complex in which RFC2 is in the front. B, views from the DNA-interacting pore of the clamp loader subunits. Domains I and II of AAA⁺ domain and α14 and α15 of RFC2 are indicated.

RFC2 monoubiquitylation in response to DNA-damaging agents is RAD18-dependent. A, lysates from RFC2-HA- and FLAG-ubiquitin-co-transfected 293A cells were analyzed by immunoprecipitation and Western blotting. pCDNA3·RFC2-HA was co-transfected either with pCAGGS-FLAG-Ubiquitin (lanes 4 and 8) or empty vector (lanes 3 and 7) in 293A cells. The following day, cells were treated with MMS for 8 h, and then cell extracts were recovered. Cell extracts were immunoprecipitated with anti-RFC2 antibody. The resulting immune complexes were recovered using protein A/G-agarose and detected by immunoblotting with anti-RFC2 antibody (lanes 1–4) or anti-FLAG antibody (lanes 5–8). Asterisks show nonspecific bands. B, Western blot of lysates from 293A cells overexpressing hRAD18. pCDNA3·RFC2-HA was co-transfected either with pCAGGS·hRAD18 (lane 3) or empty vector (lane 2) in 293A cells. Chromatin fractions were prepared and analyzed by Western blotting with anti-RFC2 (lower panel) or anti-PCNA (upper panel). The arrowheads indicate the position of molecular mass markers (kDa). C, Western blot of lysates from HCT116 cells (WILD) or RAD18-deficient HCT116 cells (RAD18^-/-). HCT116 cells transfected either with empty vector or pCAGGS·hRFC2 were treated with 0.85 mm MMS for 8 h. Chromatin fractions from the resulting cells were analyzed by immunoblotting with anti-RFC2 antibody. The arrowheads indicate the position of molecular mass markers (kDa). I.P., immunoprecipitate; I.B., immunoblot; DMSO, Me₂SO; Ub, ubiquitin.

In vitro monoubiquitylation of RFC2. In vitro ubiquitylation was carried out by mixing RFC with mouse E1, RAD18-RAD6A complex, ubiquitin, and singly primed single stranded M13 mp18 DNA in the presence or absence of RPA or PCNA as indicated. The reaction products were analyzed by Western blotting with anti-RFC2 or anti-PCNA antibody.