14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

Abstract

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.

Fig. 1

hEXO1 interacts with 14-3-3 proteins. (A) Yeast two hybrid assay demonstrating specific interaction between 14-3-3 isoforms and hEXO1. S. cerevisiae EGY48/pSH18-34 was transformed with pEG-hEXO1 (upper panel) or pEG-Raf-1 (lower panel) along with pJG vectors that express WT 14-3-3ζ, its mutant derivatives, or other 14-3-3 isoforms as indicated. The production of blue color reflects positive interactions. (B) GST pulldown assay between recombinant GST-14-3-3 proteins and radioactive labeled IVTT hEXO1, either S-tagged, His-tagged or non tagged, showing distinct interaction between hEXO1 and six of the seven 14-3-3 isoforms and non interaction in control reactions with GST or beads (right lanes). (C) GST pulldown between recombinant GST-14-3-3 proteins and purified hEXO1 detected by WB against hEXO1. Positive interaction is demonstrated for 14-3-3η. (D) GST pulldown assay between recombinant GST-14-3-3 proteins β, γ, η and σ and radioactive labeled IVTT proteins hEXO1, MSH2, MSH6, MLH1 and PMS2, showing specific interaction only for hEXO1.

Fig. 2

hEXO1 excision activity on blunt end DNA is affected by 14-3-3. hEXO1 nuclease activity assayed on 5’ radioactive labeled 42-mer homoduplexes by incubation with recombinant purified hEXO1, non-tagged or His tagged. (A) hEXO1 nuclease activity assayed in the presence of excess 14-3-3. In absence of hEXO1 or in the presence of 14-3-3 no degradation of substrate occurs. The substrate was between 10% and 25% degraded and separated on 12% denaturing polyacrylamide gel. (B) Nuclease activity of hEXO1 on 5’labeled substrate after addition of 14-3-3 isoforms. Values are expressed in percentage excision of substrate by hEXO1 in presence of 14-3-3. The hEXO1 is set as control to normalize the addition the 14-3-3 isoforms. Excision activity is of hEXO1 on the substrate is significantly stimulated by 14-3-3η and σ (mean ± SD.; *p = 0.0158; ***p = 0.0009). The control reaction with GST-14-3-3ε (lane 11) demonstrates the purity of the GST-14-3-3 preparations. (C) hEXO1 nuclease activity assayed in the presence of excess recombinant His-14-3-3ζ or the binding deficient form His-14-3-3ζ K49E(2-18 fold molar excess). Activities are defined as product band proportion of total band intensity and data given as mean ± SD from 4 independent experiments. (D) Double GST-pulldown assays in which IVTT hEXO1 and 14-3-3ζ (bovine) were added separately (lanes 4-5, 7-8) or in combination, simply added the reaction tubes simultaneously (lanes 5 and 9) or pre-incubated and then added (lanes 6 and 10).

Fig. 3

In vitro DNA replication is unaffected by addition of recombinant hEXO1. (A) In vitro DNA replication assay showing the ability of pi 86 to confer autonomous replication activity to non-origin plasmid XL-Topo. DpnI digests methylated (ii) but not unmethylated (i) pBluescript. HeLa cell extracts are incubated with XL-Topo or XL-Topo containing the p 186 minimal region of the Ors8 origin of DNA replication and either left untreated (−) or digested with indicated amounts of DpnI (+). Input methylated DNA is digested by DpnI whereas newly replicated DNA becomes resistant to digestion, as shown by the absence of digestion products (iii). (B) In vitro DNA replication assay showing no effect of recombinant hEXO1, 14-3-3ε, or both components on in vitro replication of XL-Topo containing p186. The arrows indicate the relaxed circular (form II) and linear (form III) plasmid DNA. Form I (supercoiled plasmid DNA) overlaps with the region of the DpnI digestion products, and is not detectable in the figure. Quantification of the Dpn1-resistant forms Hand III DNA is shown at the bottom of panel B, as percentage over the total DNA.

Fig. 4

Mapping of 14-3-3 binding motif in hEXO1 by GST pull-down assays. (A) Primary amino acid sequence of hEXO1 showing the N and I nuclease domains (grey), phosphorylated amino acids (red, *HU induced), known sequence motifs; NLS (blue), MIP-box (pink), PIP-box (yellow), and predicted 14-3-3 binding motifs (turquoise) highly resembling a mode 1 sequence peptide (RSxpS/TxP). Underlined residues (348-548) represent the 14-3-3 interaction domain on human Exo1 suggested by Co-IP data. (B) GST-pull-down assay between GST-14-3-3 isoforms and IVTT hEXO1 WT and the 7mut variant showing reduced interaction in comparisons. The 7mut is alterated at S422A, S454A, S598A, T621A, S714A, S674A and S746A. (C) GST-pulldown assay between GST-14-3-3 isoforms and selected IVTT hEXO1 variants. The triple variant 3HU is mutated specifically at the HU induced sites: S454A, T621A and S714A. Control reactions with MMR proteins GST-MSH2 and GST-MLH1 demonstrates functional binding ability and protein stability of the separate variants. The table on the right indicates the relative binding affinities. Band intensities were estimated with ImageQuantTL software. Interaction affinities for the variants are estimated relative to WT interaction, which is set to be 100% for each interacting protein. (D) GST-pulldown assay between GST-14-3-3β and IVTT hEXO1 WT, S746A and S746D, in the absence or presence of 14-3-3 inhibitor peptide R18. Binding to GST-MLH1 was included as a control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5

hEXO1 phosphorylation variants co-localize with PCNA in the nucleus. (A) HeLa cells transiently expressing YFP-hEXO1 (WΓ or variant forms) visualized with confocal laser-scanning microscopy. The top row shows the YFP signal, while the lower panel represents the Nomarski view showing whole cells and nuclear YFP protein distribution. Variants include YFP-hEXO1 S422A, S422D, 4S4D: S422D, S598D, S714D and S746D, 6mut: S422A, S454A, S598A, T621A, S714A and S746A and 7mut: S422A, S454A, S598A, T621A, S714A, S674A and S746A. (B) NIH-3T3 cells co-expressing CFP-PCNA (green) and YFP-hEXO1 (red, WT or variant forms) visualized with confocal laser-scanning microscopy. Yellow represents the overlay demonstrating protein co-localization. (C) GST-pulldown assay between GST-Importin β/α3 and IVTT hEXO1 WT, S422A and S422D in the presence of increasing amounts of recombinant His-14-3-3ζ. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6

Potential functional outcomes of hEXO1-14-3-3 association in the DDR. Model of three plausible outcomes hEXO1/14-3-3 association presumed to indirectly regulate hEXO1 excision activity upon DNA damage induction. (A) Extensive 5′ → 3′ DNA resection by hEXO1 at the DNA damage site is possibly hindered by protein phosphorylation and subsequent binding by 14-3-3 proteins, mediating its ubiquitination and degradation. (B) Active hEXO1 at the DNA damage site is initially phosphorylated or otherwise modified which enables 14-3-3 binding. Chk1 is also phosphorylated in response to DNA damage and associates specifically with 14-3-3 proteins. 14-3-3 scaffolding of Chk1 and hEXO1 could mediate further phosphorylation of hEXO1 by Chk1 inhibiting its activity and preventing excessive DNA resection at the damage site. (C) Binding of hEXO1 by 14-3-3 might be related to inhibition of its apoptotic signaling ability, either mediated by nuclear export or altered protein affinity towards other binding partners in the apoptotic pathway.