High mobility of flap endonuclease 1 and DNA polymerase eta associated with replication foci in mammalian S-phase nucleus

Abstract

Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase eta (Pol eta). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Pol eta and GFP-Fen1 are mobile with residence times at RFi (t(m)) approximately 2 and approximately 0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and t(m) increased, but it then recovered. The mobilities of focal GFP-Pol eta and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks.

Figure 1.

Expression of GFP-tagged human Fen1 in transfected Chinese hamster V79 cells. (A) Two patterns of GFP-Fen1 distribution in the nuclei of transiently transfected live cells: diffuse (left) and focal (right). Bar, 10 μm. (B) Western blots of total protein from stably transfected V79 cells probed with antibodies against human recombinant Fen1 (right) or GFP (left). Endogenous Fen1 protein is marked as e-Fen1.

Figure 2.

Evidence of interaction of GFP-Fen1 protein with endogenous PCNA in Chinese hamster cells. (A) Immunoprecipitation of endogenous PCNA from cell lysates of V79 cells stably expressing GFP-Fen1 with antibodies against human recombinant Fen1 (hr-Fen1, lane 1), GFP (lane 3), or Fen1 peptide (lane 5). As controls, affinity-purified antibodies against CSB protein (lane 2), CSA protein (lane 4) or preimmune serum (lane 6) were used. (B) Comparative PCNA immunoprecipitation with antibodies against GFP (lane 2) and Fen1 peptide (lane 3). Lane 1 shows input. All blots were probed with antibodies against PCNA. (C) Colocalization of endogenous PCNA foci and GFP in V79 cells transiently transfected with GFP-Fen1 plasmid and preextracted with CSK buffer containing 0.5% Triton X-100. Bar, 10 μm.

Figure 3.

Expression of GFP-CSA protein in human cells (CS3BESV) with mutation of endogenous CSA protein. (A) Images of cells expressing GFP-CSA protein. Left two images show transiently (top) or stably (bottom) transfected living cells, and right two images show unirradiated (top) or UV-irradiated (bottom) stably transfected cells preextracted with CSK buffer containing 0.5% Triton X-100 (Kamiuchi et al., 2002). Bar (A), 10 μm. UV dose was 34 J/m² followed by 1-h incubation of cells in growth medium. (B) Survival of stably transfected GFP-CSA expressing cells (clone OS-7) after UV, parental cells (CS3BESV), and normal human fibroblasts (line WI38VA13). (C) Immunoblot of proteins from OS-7 cells probed with antibodies against GFP. (D) UV-induced insolubilization of GFP-CSA protein in OS-7 cells and its stimulation by pretreatment with trichostatin A (300 nM, 24 h). Roscovitine (20 μM) was added for 6 h before UV-irradiation. Mean GFP-CSA fluorescence intensity >50 of nuclei of OS-7 cells was measured under identical conditions (amplification gain and magnification) for all variants.

Figure 4.

FRAP analyses of mobility of GFP-Fen1 in transiently transfected (A and B) or stably expressing cells (C and D) and GFP-Polη in transiently transfected cells (E and F). Bar (A, C, and E), 5 μm; numbers show seconds after photobleaching. In B, D, and F, vertical bars represent SEs of average values from 10 to 30 cells for each variant. Bleached segments are boxed.

Figure 5.

FRAP assay of mobility of GFP-CSA in transiently transfected V79 (A) or stably expressing OS-7 cells (B and C). Vertical bars in A show SE; numbers in B show seconds after photobleaching. Bar (B), 10 μm; bleached segment is boxed. Redistribution times in C were calculated from the original FRAP curves as mean times for recovery of 50–75% of the maximal fluorescence recovery ± SD. Trichostatin A (TrA; 300 nM) was added for 24 h, UV-dose was 34 J/m² followed by 1-h incubation in growth medium.

Figure 6.

MMS-induced changes of mobility of GFP-Fen1 in stably expressing V79 cells. In A, bleached segment is boxed; bar is 5 μm, and numbers show seconds after bleaching. Vertical bars in B show SE of average values for 10–30 cells, results obtained at 2–3, 5–6, and 8–9 h after MMS treatment are combined. (C) Mobile fractions of GFP-Fen1 (circles) or GFP-Polη (triangles) at different times after MMS calculated as described in Materials and Methods. (D) Changes of residence time of GFP-Fen1 at replication foci after MMS, calculated as described in Materials and Methods.

Figure 7.

Mobility of GFP-Fen1 after treatment of transiently transfected V79 cells with bleomycin (A) and survival of V79 cells after bleomycin (B). Treatment with bleomycin in B was for 2 h. Vertical bars in A show SE from 10 cells; vertical bars in B show SD from three experiments.

Figure 8.

Mobility of GFP-Polη at different times after MMS treatment (A and B), mobility of GFP-PCNA 5 h after MMS (C and D), and induction of PCNA monoubiquitination by MMS (E). In A–D, treatment was for 1 h with 0.03% MMS, and in E 0.01% MMS was added for 1 h and after washing cells were incubated in growth medium for 5 h. Bar (C), 10 μm. Vertical bars in A show SE calculated for 10–30 cells. Residence times in B were calculated as described in Materials and Methods. Western blot in E was probed with mouse monoclonal antibodies against PCNA (clone PC-10); PCNA means endogenous PCNA.