Molecular basis for assembly of the shieldin complex and its implications for NHEJ

Shieldin, including SHLD1, SHLD2, SHLD3 and REV7, functions as a bridge linking 53BP1-RIF1 and single-strand DNA to suppress the DNA termini nucleolytic resection during non-homologous end joining (NHEJ). However, the mechanism of shieldin assembly remains unclear. Here we present the crystal structure of the SHLD3-REV7-SHLD2 ternary complex and reveal an unexpected C (closed)-REV7-O (open)-REV7 conformational dimer mediated by SHLD3. We show that SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 by forming β sheet sandwich. Disruption of the REV7 conformational dimer abolishes the assembly of shieldin and impairs NHEJ efficiency. The conserved FXPWFP motif of SHLD3 binds to C-REV7 and blocks its binding to REV1, which excludes shieldin from the REV1/Pol ζ translesion synthesis (TLS) complex. Our study reveals the molecular architecture of shieldin assembly, elucidates the structural basis of the REV7 conformational dimer, and provides mechanistic insight into orchestration between TLS and NHEJ.

The crystal structure presented here reveals the molecular assembly of the shieldin complex. Interestingly, the structure captures C (closed)-REV7-O (open)-REV7 dimer. This is stabilized by SHLD3 that wraps across the dimer using its REV7 binding motifs (RBMs). The authors found that SHLD2 facilitates the interaction between O-REV7 and the N-terminus of SHLD3 by forming a beta sheet insertion between them. The authors show that the conformational dimer seen in the crystal structure is critical for an efficient NHEJ activity. By overlaying the current structure with previously published REV3-C-REV7-REV1 structure, the authors find that the SHLD3 binding inhibits REV1 polymerase from binding to REV7, thus revealing exclusivity of NHEJ and REV1mediated TLS. Furthermore, they have shown that the SHLD3-mediated REV7 dimer could bind to REV3 and may act as protein binding platform. Although the overall resolution of the crystal structure (3.5 A) is moderate, the author have validated key interfaces by diligently making mutations and studying their impact on protein-protein interaction by ITC, MALS and gel-filtration assays. They also complemented their biophysical observation with NHEJ functional assays and cell measurements. The authors should address the following points. Points.
1) The dimer of tetramer seen in the crystal structure ( Figure 1f) and MALS (Supplementary Figure  2b) may be a concentration dependent assembly that may then only exist at higher concentrations. If this is a biologically relevant assembly making mutations at the interface should have an effect (e.g. NHEJ efficiency?). It would be helpful to test this with appropriate mutations. 2) Is the heterogeneity in the molecular weight observed in MALS data (Supplementary Figure 3b) for REV7wt-SHLD3 (1-82) because SHLD2 is missing from the experiment? As there is indication from MD simulation showing SHLD2 may help form a more stable complex, what happens to the molecular weight distribution (WT and mutants in supplementary figure 3) when you add SHLD2? 3) Fix typo line 164-FXPWFP instead of PXPWFP. 4) For completion, in Figure 4, could SHLD3 (when not bound to REV7) by itself interact with REV1? 5) While it is admirable to use gel-filtration assay to see the formation of complexes between different components, given the low resolution of Superdex200 column (in present case), it is hard to interprete the results. Ideally, these could be complemented with in vitro pulldowns using MBP tag on the SHLD2 (e.g. Figure 5)? It's just a suggestion.
6) The line 268 should read …Interestingly, the structural alignment ( Figure 6c) also shows beta1 of SHLD2 occupies the position of RBM2 of SHLD3 (as this is concluded from structural alignment of C-REV7-SHLD3 and O-REV7-SHLD2). 7) There were several different proteins and mutants expressed as part of this manuscript, were all of them purified using the same methods and buffer given in the general protein expression section of the methods? Addressing this would help with future reproducibility issues. 8) Were ITC experiments repeated? One would like to see a standard deviation for the measured binding affinity values.

Point-by-point Responses
"Molecular basis for assembly of the shieldin complex and its implications for NHEJ" (NCOMMS-19-1124676-T) Point-by-point responses to the comments by Reviewer #1: The work done by Liang et al. described the crystal structure of the Shieldin complex, and investigated its function in NHEJ. While this study is overall interesting, the results they presented are lack of clarity. 1) Whereas the quality of the electron density of the whole molecule is good, the local density at regions that are important for the formation of complex should be shown. Otherwise, it is hard to adjust the accuracy of the structure.
Based on the reviewer's suggestion, we have included the local density at regions that are important for the formation of the complex in Supplementary Fig. 1, which is also shown below ( Figure R1).  3) It is more problematic that the assembly status of the samples of REV7WT-SHLD3 are quite heterogeneous, as shown in Fig. S3b. This is also true for Fig. S2b. This issue is not discussed until Fig. 5a, in which the target complex turns out not to be a major species in solution. The mixture nature of the samples should be discussed and clarified, and the accuracy of the molecular weights measured by MALS should be indicated.
To clarify the mixture nature of the samples, we revised the manuscript as  Table 1)'. In the revised figures, the accuracy of the molecular weights measured by MALS has been indicated by the fitted errors obtained from the data analysis software, which are showed in the brackets. We also calculated the error of observed masses compared with calculated masses (Table R1).  Fig. 2b are simply reversed.

4) The colors for the positive-and negative charges in
We are afraid that the reviewer has misunderstood Fig. 2b. We want to clarify that the residues shown in sticks on the surface are not residues of the electrostatic model. To avoid potential misunderstanding, we added following highlighted description in the revised figure legends. In left panel, C-REV7 is shown as electrostatic surface model and the labelled amino acid residues of O-REV7 that interact with C-REV7 are shown in sticks. In the right panel, O-REV7 is shown in electrostatic surface model while labelled residues of C-REV7 that interact with O-REV7 are shown in sticks.

5) It is interesting that the authors found
). Yet, the reasoning for these observation is not clearly stated, as the data and the discussion on the asymmetry are blended with other data.
We have added the following highlighted description in the revised version. This is because Lys129, Lys190 of C-REV7 locates at the asymmetric interface of C-REV7-O-REV7 and contributes to the interaction while Glu35, Lys44 of C-REV7 diverge from the interface and have no effect on the interaction.

6) The authors discuss quite extensively that SHLD3(1-37) contributes limited impact to the formation of C-REV7-O-REV7
dimer. Yet, no figure is used to show the structure of SHLD3(1-37). An expanded Fig. 3a to include the whole SHLD3 molecule may be suitable for this end.
As the reviewer suggested, we expanded Fig. 3a to include the whole SHLD3(1-37) to show its structure in the revised version (Fig. R3). Figure 3a) The interface between SHLD3 and C-REV7-O-REV7. C-REV7 and O-REV7 are shown in electrostatic surface representation (positive potential, blue; negative potential, red), SHLD3 in ribbon view, and the FXPWFP motif in sticks. The N terminus of SHLD3 is also indicated. Residues 1-37 of SHLD3 are shown in ribbon.

Point-by-point responses to the comments by Reviewer #2:
Cells are tribulated with endogenous and exogenous genotoxins that result in damaged-DNA. These damages are mainly repaired by two major pathways: 1) non-homologous end-joining (NHEJ) and homologous recombination (HR). The pathway choice for repair is considered one of the key factors that determines the fate of the cells. Moreover, there is increasing interest in some advanced cancer drugs (e.g. PARP inhibitors) that target defects in DNA repair pathways (e.g. HR) in cells. One resistance mechanism for PARP inhibitors in BRCA1-deficient cells is the loss of 53BP1-RIF1-REV7-shieldin pathway that downregulates NHEJ as shielding complex protects DNA breaks from resection. Also, shieldin proteins interact with translesion synthesis complex (involving REV1), which may provide additional resistance routes for cancer cells to escape the treatment. Thus, the ternary crystal structure of components (REV7-SHLD2-SHLD3) of the shielding complex described by Liang et al is timely and of broad interest for understanding resistance and potential therapeutic opportunities in the DNA damage repair-based cancer therapy.

This is stabilized by SHLD3 that wraps across the dimer using its REV7 binding motifs (RBMs). The authors found that SHLD2 facilitates the interaction between O-REV7 and the N-terminus of SHLD3 by forming a beta sheet insertion between them. The authors show that the conformational dimer seen in the crystal structure is
critical for an efficient NHEJ activity. By overlaying the current structure with previously published REV3-C-REV7-REV1 structure, the authors find that the SHLD3 binding inhibits REV1 polymerase from binding to REV7, thus revealing exclusivity of NHEJ and REV1-mediated TLS. Furthermore, they have shown that the SHLD3-mediated REV7 dimer could bind to REV3 and may act as protein binding platform. Although the overall resolution of the crystal structure (3.

A) is moderate, the authors have validated key interfaces by diligently making mutations and studying their impact on protein-protein interaction by ITC, MALS and gel-filtration assays. They also complemented their biophysical observation with NHEJ functional assays and cell measurements. The authors should address the following points.
We would like to thank the reviewer for these positive comments. We have addressed each of the reviewer's comments as detailed below. Points.

1) The dimer of tetramer seen in the crystal structure (Figure 1f) and MALS (Supplementary Figure 2b) may be a concentration dependent assembly that may then only exist at higher concentrations. If this is a biologically relevant assembly making mutations at the interface should have an effect (e.g. NHEJ efficiency?). It would be helpful to test this with appropriate mutations.
We tried to test this. However, it is difficult to make a mutation that only disturbs the dimer of tetramer while does not affect the C-REV7-O-REV7 conformational dimer. For example, Lys190 of O-REV7 locates at the interface of the dimer of tetramer and may contributes to the formation of the dimer of tetramer. However, REV7 K190A also fails to form the C-REV7-O-REV7 conformational dimer. On the other hand, as reviewer 1 suggested, the proposed dimer of tetramer is a minor population in solution, and thus Fig. 1f seems meaningless, so we deleted it in the revised version. Figure 3b) for

3) Fix typo line 164-FXPWFP instead of PXPWFP.
We apologize for this typo. We have corrected it. Figure 4, could SHLD3 (when not bound to REV7) by itself interact with REV1?

4) For completion, in
We performed ITC experiments which showed that SHLD3 by itself could not interact with REV1 ( Figure R5). In Figure 4, we want to emphasize that REV1 interacts with C-REV7 ( Fig. 4c and 4d) and the FXPWFP motif of SHLD3 disrupts the interaction between REV1 and C-REV7 (Fig. 4b versus Fig. 4c and Fig. 4e versus Fig. 4f). To make it easier for interpreting these results, we have revised the description of Fig. 4 in the results section.

Figure R5 ITC measurement of the interaction between SHLD3
(1-82) and cREV1 CTD. This is a representative result. N.D.: no detectable binding.

5) While it is admirable to use gel-filtration assay to see the formation of complexes between different components, given the low resolution of Superdex200 column (in present case)
, it is hard to interpret the results. Ideally, these could be complemented with in vitro pulldowns using MBP tag on the SHLD2 (e.g. Figure 5)? It's just a suggestion.
6) The line 268 should read …Interestingly, the structural alignment (Figure 6c) also shows beta1 of SHLD2 occupies the position of RBM2 of SHLD3 (as this is concluded from structural alignment of C-REV7-SHLD3 and O-REV7-SHLD2).
We have revised it as the reviewer suggested. 7) There were several different proteins and mutants expressed as part of this manuscript, were all of them purified using the same methods and buffer given in the general protein expression section of the methods? Addressing this would help with future reproducibility issues.
Yes, all the proteins used in the manuscript were purified using the same methods and buffer given in the general protein expression section of the methods. To address this, we added 'All proteins used in this study were purified using the same methods and buffer as described above' in the revised version.

8) Were ITC experiments repeated? One would like to see a standard deviation for the measured binding affinity values.
We showed representative results previously. Now we have calculated the standard deviations for the measured binding affinity values from three independent experiments in the revised version 7c,7e,8a,8c,. The following Table R2 summarizes the results of repeated experiments.