Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

Abstract

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.

Figure 1.

Recruitments of EGFP-tagged proteins related to DNA repair synthesis to laser-induced DNA damage. Five EGFP-fused RFC subunits, PCNA and catalytic subunit of DNA polδ (Polδ1) were transiently expressed in HeLa cells, and a 405-nm UVA-laser (500 scans) was directed to identical sites within nucleus (white arrowheads). The picture was taken before (pre-irradiation), immediately (within 10 s) and 20, 60, 120 and 300 s after irradiation.

Figure 2.

(A) Recruitment of EGFP-tagged proteins related to DNA repair synthesis to DNA damage induced by a 365-nm UVA-laser. (A) Nuclei (arrowheads) of HeLa cells expressing EGFP-RFC140, EGFP-PCNA and EGFP-Polδ1, were irradiated with a 365-nm UVA laser as described in ‘Materials and Methods’ section. (B) Accumulation of EGFP-fusions, RFC140 (square), PCNA (triangle) and Polδ1 (circle) in (A) was measured as the fold increase of fluorescence intensity at an irradiated site. Data were taken from five independent experiments. Error bars represent standard errors. (C) Intensity at laser irradiation sites of EGFP-fusions and five RFC subunits was plotted as in (B). (D) Maximum intensity (MI) and the time to reach MI (t MAX) were represented in each GFP-fusion. A half of MI (1/2 MI) was calculated as 0.5 × (MI − 1) + 1. Thus, t 1/2 MI indicates the time to reach 50% of MI.

Figure 3.

Recruitments of endogenous RFC140 and PCNA to laser-induced DNA damage. HeLa cells were plated in glass-bottomed dishes 2 days before irradiation, and cells were irradiated with a 405-nm UVA-laser at the positions indicated between the white arrowheads on the DAPI picture. At 2, 5, 10 and 30 min after irradiation, cells were fixed with paraformaldehyde (for RFC140) or methanol followed by detergent-treatment (for PCNA) and probed with anti-RFC140 or anti-PCNA antibody described in ‘Materials and Methods’ section. Phosphorylated H2AX (γH2AX) was the marker for showing actual laser irradiation.

Figure 4.

Accumulation of EGFP-RFC140 and EGFP-PCNA in response to laser-induced DNA damage in xrcc1-deficient and xrcc1-proficient mouse embryonic cells. Nuclei of xrcc1-deficient (KO) or proficient (WT) mouse embryonic cells expressing EGFP-RFC140 or EGFP-PCNA were irradiated with a low dose (SSBs) or a high dose (SSBs + DSBs) of 365-nm UVA-laser light. Time-lapse pictures were taken as indicated in Figure 2A.

Figure 5.

Deletion analysis of RFC140. (A) The domain structure of RFC140 and RFC140 fragments. Details of each domain are described in the text. The degree of accumulation of each fragment at laser-induced DNA damage is indicated as follows: +++, accumulation of full-length protein (as 100% when 120 s after irradiation); ++, weaker accumulation of full-length (more than 70%); +, much weaker accumulation of full-length (30–70%); +/−, very weak accumulation (less than 30%) and -, no accumulation. Localization of each EGFP-fragment without 2xNLS is also indicated. Nu and Cyto indicate nuclear and cytosolic localization, respectively. (B) Accumulation of each EGFP-fragment is shown at 120 s time point after irradiation.

Figure 6.

Accumulation kinetics of a series of deletion fragments with or without F6A/F7A substitution. (A) Sequence alignment of the N-terminal portion of RFC140 orthologs from nine eukaryotes. Approximately 10 amino-acid residues are indicated. Red-colored residues are highly conserved among all species, and are indicated as the PIP-box (see text). EGFP-fused fragments 1–369 (B), 1–397 (C), 1–493 (D), 1–733 (E) and full-length (1–1147, F) having normal (closed squares) or the F6A/F7A mutation (open squares) were transiently expressed in HeLa cells and accumulation was measured as the fold increase of fluorescence intensity. Error bars indicate standard error. Error bars not indicated are smaller than symbols. Pictures represent accumulation of FA mutant fragments, and are taken before and 120 min after laser irradiation. Data were taken from five independent experiments. White arrowheads indicate the laser irradiation region.

Figure 7.

GST-pull-down assay. GST-fused RFC140 fragments were purified and indicated as asterisks on Coomassie staining gel (bottom column). GST-fragments immobilized on glutathion sepharose beads were mixed with purified His-PCNA, and GST-pull-down assay was done as described in ‘Materials and Methods’ section. On the His-PCNA lane, 10% of input protein was loaded. Bound PCNA was detected by Western blots with anti-His antibody (top column).

Figure 8.

Cellular localization of endogenous RFC140 and PCNA in asynchronous 293T cells. S1, S2 and P2 indicate cytoplasmic, nucleoplasm (soluble nuclear) and chromatin/nuclear matrix (insoluble nuclear) fractions, respectively. MEK2 and ORC2 blots are represented as a markers specific to cytoplasmic and chromatin fractions, respectively.

Figure 9.

Effect of absence of RFC140 on accumulation of PCNA at DNA damage. (A) siRNA-mediated RFC140 knock-down. Whole cell extracts were prepared from mock-, Luciferase siRNA- and RFC140 siRNA-treated HeLa cells as described in ‘Materials and Methods’ section. Blot was probed with anti-RFC140, PCNA and Actin antibodies. (B) Indirect immunofluorescence. Luciferase siRNA- and RFC140 siRNA-treated HeLa cells were irradiated with a 405-nm UVA-laser and fixed at 2 or 5 min after irradiation. Indirect immunolabeling was performed as described in ‘Materials and Methods’ section.