Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

Abstract

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.

Fig. 1

Sub-nuclear localization of human MMR proteins. NIH3T3 cells were transiently transfected with plasmid DNA and incubated for 24–48 h before fluorescent fusion proteins were visualized by confocal imaging; CFP (green), YFP (red), and co-localization (yellow). (A) CFP-PCNA and YFP-hEXO1, from the left; early S-phase cells are characterized by numerous small foci evenly distributed throughout the nucleus, mid S-phase cells are characterized by an enlargement of foci size, reduction in the number of foci and alignment of foci at the nuclear periphery, and finally late S-phase cells are characterized by few and larger foci. (B) CFP-PCNA and YFP-hMLH1; diffuse nuclear distribution of hMLH1 seen together with PCNA foci in all three stages of the cell cycle: early S-phase (left), mid S-phase (center), or late S-phase (right). (C) CFP-PCNA and YFP-hMSH2; hMSH2 forms nuclear foci co-localizing with PCNA in early (left), mid (center), and late (right) S-phase cells. (D) CFP-PCNA and YFP-hMSH6; hMSH6 forms nuclear foci co-localizing with PCNA in early (left), mid (center), and late (right) S-phase cells. The percentage of pixels overlapping (co-localizing proteins) in the presented images is displayed in the table.

Fig. 2

Recruitment of MMR proteins to micropoint laser induced DSBs. HeLa cells were transiently transfected with plasmid DNA and incubated for 24 h for expression of fluorescent fusion proteins; CFP (cyan) or YFP (green). Expressing cells were irradiated using a 435 nm micropoint laser and real time protein recruitment monitored in the relevant channels. Extended focus view of fluorescing cells obtained by Z-stack imaging 2 min after micropoint laser irradiation. The pictures represent typical targeting of 1–3 cells in one round of laser firing. Following micro-irradiation, cells were fixed, blocked, and stained with primary antibodies against XRCC1 and γH2AX combined with Alexa Flour secondary antibodies and detected with confocal fluorescence microscopy. (A) YFP-hEXO1 expressing cells targeted in a thick line using 11% laser intensity (B) CFP-PCNA expressing cells targeted in 1 or 2 thick lines using 8% laser intensity (C) CFP-hMSH2 expressing cells targeted in a thick line using 11% laser intensity (D) CFP-hMSH6 expressing cells targeted in a thick line using 11% laser intensity (E) YFP-hMLH1 expressing cells targeted in a thick line using 14% laser intensity.

Fig. 3

Nuclear foci formation and DNA double strand break recruitment of YFP-hEXO1 COOH-terminal truncated variant. (A) Consensus PIP-box, (h) is a hydrophobic residue and (a) are aromatic residues. EXO1 sequences from the following species were aligned; human (NM_130398), chimpanzee (XM_514304), dog (XP_547491), mouse (NP_036142), and rat (XP_222932). Using homology search and bioinformatic tools Jpred3 and PredictProtein putative PIP-box motifs in conserved regions were identified and indicated. (B) hEXO1 protein domain structure, regions involved in protein interactions are specified; hMSH3 (green), hMSH2 (blue), and hMLH1 (pink), nuclease domains N and I, and the nuclear localization signal (NLS). Positions of putative PIP boxes and amino acid substitutions are indicated. Amino acid substitutions were introduced with site directed mutagenesis. (C) NIH3T3 cells were transiently transfected with plasmid DNA and incubated for 24–48 h before visualization of fusion proteins by confocal imaging; CFP (green), YFP (red), and co-localization (yellow). PCNA forms foci but the COOH-terminally truncated variant is unable to form foci as seen by the diffuse nuclear staining pattern. The percentage of pixels overlapping (co-localizing proteins) in the presented image is displayed in the table. (D) HeLa cells were transiently transfected with plasmid DNA and incubated for 24 h for expression of fluorescent fusion protein YFP-hEXO1-S702X (light green). Expressing cells were irradiated using a 435 nm micropoint laser at 14% laser intensity, and extended focus view of fluorescing cells obtained by Z-stack imaging 2 min after micropoint laser irradiation. Recruited protein fluorescence at the irradiated path line is indicated by an arrow for each cell for clarification. Following micro-irradiation, cells were fixed and stained with antibodies against XRCC1 and γH2AX to confirm induction of SSBs and DSBs at the laser irradiated path line (not shown).

Fig. 4

Sub-nuclear localization and co-localization of CFP-PCNA and YFP-hEXO1 putative PIP-box variants. NIH3T3 cells were transiently transfected with plasmid DNA and incubated for 24–48 h before fusion proteins were visualized by confocal imaging; CFP (green), YFP (red), and co-localization (yellow). (A) YFP-hEXO1-Q788A;L791A from the left; YFP-hEXO1-Q788A;L791A does not form foci in early, mid or late S-phase cells (B) YFP-hEXO1-Q154A;Y157A from the left; early S-phase cells showing co-localization of foci and mid S-phase cells with a less distinct co-localization (C) YFP-hEXO1-Q285A;F288A from the left; early S-phase cells showing co-localization of foci and mid S-phase cells showing a less specific co-localization (D) YFP-hEXO1-F506A;F507A from the left; early S-phase cells with no YFP-hEXO1-F506A;F507A foci and non S-phase cells displaying cytoplasmic localization of hEXO1-F506A;F507A (red). The percentage of pixels overlapping (co-localizing proteins) in the presented images is displayed in the table (n.d.: not determined).

Fig. 5

Distribution of nuclear foci in cells co-expressing CFP-PCNA and YFP-hEXO1 variants. For each experiment 50 co-transfected cells were scored according to categories described herein. (A) Cells co-expressing hEXO1-WT and PCNA display co-localization throughout S-phase (B) Variant hEXO1-Q788A;L791A is unable to form replication foci, but expression does not affect the formation of replication foci by PCNA. (C) Cells expressing hEXO1-Q154A;Y157A display a higher proportion with no foci and early S phase foci, when compared to cells expressing hEXO1 WT, this applies for both hEXO1 and PCNA replication foci. (D) Cells expressing hEXO1-Q285A;F288A display a higher proportion with no foci and early S phase foci, when compared to cells expressing hEXO1 WT, this applies for both hEXO1 and PCNA replication foci. (E) Cells expressing the variant hEXO1-F506A;F507A did not form replication foci by hEXO1 and only very few by PCNA.

Fig. 6

Binding of hEXO1 peptides to PCNA assessed by isothermal titration calorimetry. ITC measurements of the physical interaction between purified PCNA and hEXO1 peptides p22 (aa 783–804) and p16 (aa 498–513) containing the PIP-box (QIKLNELW) and MIP-box (RSRFF) sequences, respectively. (A) Thermogram of titration of hEXO1 p22 into pure buffer (B) Thermogram and binding isotherm of titration of hEXO1 p22 into PCNA solution at 30 °C (C) Thermogram of titration of hEXO1 p16 into PCNA solution at 30 °C (D) Thermogram of titration of hEXO1 p16 into PCNA solution at 10 °C (E) Table of thermodynamic parameters obtained for different PIP-box containing peptides for comparisons.

Fig. 7

Mapping of region on hEXO1 required for recruitment to laser induced DSBs. (A) Map of hEXO1 indicating protein interaction regions and schematic overview of truncation and deletion variants (I) YFP, (II) YFP-hEXO1, (III) YFP-hEXO1-S702X, (IV) YFP-hEXO1-C-term, (V) YFP-hEXO1-Δ406–507, (VI) YFP-hEXO1-Δ423–507 (VII) YFP-hEXO1-S702X; Δ423–507, (VIII) YFP-405–507. (B) HeLa cells were transiently transfected with plasmid DNA and incubated for 24 h for expression of fluorescent fusion proteins; (II) YFP-hEXO1, (III) YFP-hEXO1-S702X, (IV) YFP-hEXO1-C-term, (V) YFP-hEXO1-Δ406–507, (VI) YFP-hEXO1-Δ423–507 (VII) YFP-hEXO1-S702X; Δ423–507, (VIII) YFP-405–507. Expressing cells were irradiated using a 435 nm micropoint laser at 8–11% laser intensity, and extended focus view of fluorescing cells obtained by Z-stack imaging 2 min after micropoint irradiation. Recruited protein fluorescence at the irradiated path line is indicated by an arrow for each cell for clarification. The pictures represent typical targeting of 1–3 cells in one round of laser firing. Following micro-irradiation, cells were fixed and stained with antibodies against XRCC1 and γH2AX to confirm induction of SSBs and DSBs at the laser irradiated path line (not shown).

Fig. 8

Recruitment of hEXO1 variants to laser induced DSBs. (A) Map of hEXO1 indicating protein interaction regions and sites of amino acid variant introduction (I) YFP-hEXO1-Q154A;Y157A, (II) YFP-hEXO1-D173A, (III) YFP-hEXO1-Q285A;F288A, (IV) YFP-hEXO1-K418A, (V) YFP-hEXO1-F506A;F507A, (VI) YFP-hEXO1-Q788A;L791A (B) HeLa cells were transiently transfected with plasmid DNA and incubated for 24 h for expression of fluorescent fusion proteins; (I) YFP-hEXO1-Q154A;Y157A, (II) YFP-hEXO1-D173A, (III) YFP-hEXO1-Q285A;F288A, (IV) YFP-hEXO1-K418A, (V) YFP-hEXO1-F506A;F507A, (VI) YFP-hEXO1-Q788A;L791A, (VII) YFP-hEXO1-Q788A;L791A;L794A;W795A A;L794A;W795A. Expressing cells were irradiated using a 435 nm micropoint laser at 14% laser intensity, and extended focus view of fluorescing cells obtained by Z-stack imaging 2 min after micropoint irradiation. Recruited protein fluorescence at the irradiated path line is indicated by an arrow for each cell for clarification. The pictures represent typical targeting of 1–3 cells in one round of laser firing. Following micro-irradiation, cells were fixed and stained with antibodies against XRCC1 and γH2AX to confirm induction of SSBs and DSBs at the laser irradiated path line (not shown).