Mortusewicz O , Rothbauer U , Cardoso MC , Leonhardt H .

	Figure 1. Immunochemical detection of DNA repair intermediates after laser microirradiation. Widefield fluorescence images of HeLa cells are shown. Cells were fixed ∼5 min after laser microirradiation. Arrows mark sites of irradiation. (A) Laser microirradiation with 405 nm results in local generation of DSBs and SSBs detected by antibodies against γ-H2AX and PAR, respectively. Accumulation of endogenous PCNA can be observed at these sites. (B) Both, DNA Ligase I and III accumulate at sites of DNA damage and colocalize with PCNA. (C) XRCC1 and PCNA accumulate at laser-induced DNA damage sites. Scale bars, 5 μm.
	Figure 2. Schematic outline of the structure of DNA Ligase I and III and of fusion proteins used in this study. The catalytic core (highlighted in grey shading) is highly similar in both Ligases. The relative position of the conserved lysine residue (K) in the catalytic center is indicated.
	Figure 3. Recruitment kinetics of DNA Ligase I and III at DNA damage sites in living cells. Live cell imaging of microirradiated HeLa cells coexpressing various combinations of GFP- and RFP-tagged DNA Ligase I, DNA Ligase III, PCNA and XRCC1. For determination of the recruitment kinetics the relative fluorescence intensity at the irradiated spot was calculated and plotted as a function of time. The displayed curves were generated after integrating data from at least eight independent experiments. Error bars represent the SD. (A and B) Accumulation of DNA Ligase III at DNA repair sites precedes accumulation of DNA Ligase I (Supplementary Fig3video1). (C and D) Accumulation of RFP-PCNA can be observed as early as 2 s after irradiation, while DNA Ligase I accumulates with a delay of ∼30–60 s (Supplementary Fig3video2). (E and F) Immediate and fast recruitment of GFP-XRCC1 and DNA Ligase III to DNA damage sites (Supplementary Fig3video3). Scale bars, 5 μm. The table in (G) summarizes the kinetic parameters of PCNA, DNA Ligase I, XRCC1 and DNA Ligase III recruitment. Mean values of at least eight different cells are shown.
	Figure 4. Release kinetics of DNA Ligase I and III at DNA repair sites. Microirradiated HeLa cells were followed up several hours with a time interval of 5 min. Maximum projections of 10–12 z-Stacks were collected and the fluorescence intensities at the irradiated sites calculated and plotted as a function of time.
	Figure 5. Recruitment of DNA Ligase I to DNA damage sites is mediated by PCNA. Recruitment kinetics were determined as described in Figure 3. (A) Live cell imaging of a microirradiated HeLa cell coexpressing GFP-DNA Ligase I ΔPBD and RFP-Ligase I. Deletion of the PBD in GFP-DNA Ligase I ΔPBD almost completely abolishes recruitment to sites of DNA damage, whereas RFP-Ligase I accumulates at these sites as seen in Figure 3 (arrow). (C) A HeLa cell coexpressing DNA Ligase I PBD-GFP and RFP-Ligase I shows accumulation of both, RFP-Ligase I and DNA Ligase I PBD-GFP, at sites of microirradiation (arrows). Times after microirradiation are indicated. Scale bars, 5 μm. (B and D) Recruitment kinetics of fluorescence-tagged proteins at microirradiated sites. (E) Coimmunoprecipitations were performed in 293T cells coexpressing different combinations of RFP and GFP fusion constructs. For interaction mapping the same deletion constructs as in A–D were used. Immunoprecipitations were performed with an antibody against GFP. Precipitated fusion proteins were then detected with specific antibodies against RFP and GFP on western blots. RFP-PCNA coprecipitated with GFP-Ligase I but not with GFP-Ligase III (left panel). RFP-PCNA was also coprecipitated with Ligase I PBD-GFP but not with GFP-Ligase I ΔPBD (right panel).
	Figure 6. Recruitment kinetics of the PCNA interacting proteins DNA Ligase I and FEN-1 are similar. The structure of the fusion proteins is depicted in Figure 2. (A) Live cell imaging of a microirradiated HeLa cell coexpressing GFP-FEN-1 and RFP-Ligase I. Both FEN-1 and DNA Ligase I accumulate at DNA repair sites (arrow) with similar kinetics. Times after microirradiation are indicated. Scale bars, 5 μm. (B) Recruitment kinetics of fluorescence-tagged proteins at microirradiated sites.
	Figure 7. Deletion of the ZnF motif does not abolish recruitment of DNA Ligase III. Recruitment kinetics were determined as described in Figure 3. (A) Live cell imaging of a microirradiated HeLa cell coexpressing GFP-DNA Ligase III ΔZnF and RFP-Ligase III which accumulate at DNA repair sites (arrow). Times after microirradiation are indicated. Scale bars, 5 μm. (B) Recruitment kinetics of GFP-DNA Ligase III ΔZnF and RFP-Ligase III at microirradiated sites.
	Figure 8. Recruitment of DNA Ligase III to DNA damage sites is mediated by XRCC1. Recruitment kinetics were determined as described in Figure 3. (A) Live cell imaging of a microirradiated HeLa cell coexpressing GFP-N-DNA Ligase III ΔBRCT, containing an additional SV40 NLS and RFP-Ligase III. Deletion of the BRCT domain in GFP-N-DNA Ligase III ΔBRCT abolishes recruitment to sites of DNA damage, whereas RFP-Ligase III accumulates at these sites as seen in Figure 3 (arrow). (B) A HeLa cell coexpressing GFP-Ligase III BRCT and RFP-Ligase III which both accumulate at sites of microirradiation (arrows). Times after microirradiation are indicated. Scale bars, 5 μm. (C and D) Recruitment kinetics of fluorescence-tagged proteins at microirradiated sites. (E) Deletion of the BRCT domain results in cytoplasmic localization of the fusion protein. (F) Coimmunoprecipitations were performed with 293T cells coexpressing RFP-XRCC1 and GFP-Ligase I or GFP-Ligase III, respectively. For interaction mapping the same deletion constructs as in A–D were used. Immunoprecipitations were performed with an antibody against GFP. Precipitated fusion proteins were then detected with specific antibodies against RFP and GFP on western blots. RFP-XRCC1 was coprecipitated with GFP-Ligase III but not with GFP-Ligase I (left panel). RFP-XRCC1 was also coprecipitated with GFP-Ligase III ΔBRCT but not with GFP-N-Ligase III ΔBRCT (right panel).
	Figure 9. Model for selective targeting of DNA Ligase I and III to DNA replication and different repair pathways. All DNA Ligases use the same catalytic mechanism and show high sequence similarity in the catalytic core (grey shading). The active site lysine residue (K) in the center of the catalytic domain is directly involved in the ligation reaction. However, DNA Ligases have non-overlapping functions in DNA repair and replication and are not interchangeable. We could show that DNA Ligase I and III are targeted to different repair pathways through their regulatory PBD and BRCT domains which mediate interaction with PCNA and XRCC1, respectively. This selective recruitment of specialized DNA Ligases may accommodate the specific requirements of different repair pathways and thereby enhance repair efficiency.

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