Structure of the ZRANB3 HNH domain

To gain structural insight into the ZRANB3 HNH domain, we solved the crystal structure of the C-terminal region of the ZRANB3 (residues 948-1067) by experimental phasing. The HNH structure contains two HNH molecules in the asymmetric unit that are structurally similar, with an RMSD of 0.76 Å over 117 equivalent Cα atoms. Each HNH molecule contains one two-stranded anti-parallel β-sheet (β1–β2) surrounded by eight helices (α1–α8). The structure adopts a ββα fold16, typically found in the HNH protein family. It also contains a non-catalytic zinc-finger coordinated with four cysteine residues (Fig. 1a and Supplementary Fig. 1), conserved in a subset of HNH proteins containing the C-x-x-C dyad14. A structural homology search using the DALI server17 identified the closest structural homologues of the ZRANB3 HNH domain as phage T4 endonuclease VII, a Holliday junction-specific resolvase (PDB code 1EN7; ref. 18), and the HNH nicking endonuclease from Geobacter metallireducens (PDB code 4H9D; ref. 19) (Fig. 1b). Interestingly, sequence comparisons with other zinc-finger containing HNH proteins revealed the presence of the HNH insert (Fig. 1c), specific to ZRANB3 and the self-standing HNH proteins found in Acidobacterium bacterium and Solibacter usitatus, but absent from other HNH proteins. Overlays of the ZRANB3 HNH structure with its closest structural homologues showed that this ZRANB3-specific HNH insert adopts a well-structured α-helical domain (Fig. 1a,d). Deletion of this domain led to the loss of ZRANB3 endonuclease activity (and a reduction, but not a complete loss of the ATPase activity, Fig. 1e and Supplementary Fig. 2a; Table 2), raising a possibility that it might be important for the structure-specific recognition of the DNA substrate.

Table 1 Summary of crystallographic statistics. Full size table

Table 2 Average rates of ATP hydrolysis determined by an NADH-coupled assay according to ref. 59. Full size table

DNA binding surface

Analysis of the electrostatic surface potential of the HNH domain revealed a predominantly electropositive face (Fig. 2a), with a continuous positively charged region encompassing helices α1 and α8, as well as helices α2 and α3 in the ZRANB3-specific helical domain (Fig. 2b). Overlay of the HNH and T4 endonuclease VII structures showed that helix H2 in T4 endonuclease VII, which penetrates into the Holliday junction and stabilizes the separation between exchanging DNA strands, overlaps with the helix α8 in the HNH structure (Fig. 2c), suggesting a potential involvement of helix α8 in DNA binding. To address the role of the positively charged regions of the HNH domain, we introduced a number of mutations targeting the ZRANB3-specific helical region and helix α8 (Fig. 1c). Some of these mutations resulted in reduced DNA binding by the HNH domain (Supplementary Fig. 3b), and had a pronounced effect on the ZRANB3 endonuclease activity- as observed with the K1046A, R1048A double mutant in helix α8, and with the R1009A mutant in the ZRANB3-specific helical domain (Fig. 2d,e). Interestingly, the K1046A, R1048A double mutant retained efficient ATPase activity (Fig. 2e, Table 2), suggesting that the impact of the mutations on the endonuclease function was not linked to the ability to hydrolyse ATP. The DNA binding surface therefore likely involves helix α8 and certain positively charged regions within the ZRANB3-specific helical domain.

Characterization of the active site

Analysis of the electrostatic surface potential also revealed the presence of a negatively charged patch on a predominantly electropositive face of the HNH molecule (Figs 2a and 3a). Interestingly, overlay of the HNH and T4 endonuclease VII structures showed that the observed electronegative patch in HNH overlaps with the Mg2+ ion binding pocket in the T4 endonuclease VII active site (Fig. 3a). To gain insight into the architecture of the HNH catalytic site, we further analysed the structure of the ZRANB3 HNH domain superimposed to that of the inactive T4 endonuclease VII (N62D) in complex with the Holliday junction DNA substrate. The active site of T4 endonuclease VII adopts a characteristic ββα-metal fold, and contains a Mg2+ ion and catalytically essential residues Asp40, His41 and Asn62 (refs 20, 21, 22) (Fig. 3b). The Mg2+ ion is coordinated by the side chains of Asp40 and Asn62Asp, two oxygen atoms of the scissile phosphate and a water molecule22. A catalytic mechanism involves His41, which likely acts as a general base and activates water for nucleophilic attack. On the other hand, the Mg2+ ion is thought to stabilize the phosphoanion transition state and facilitate product formation. Although the structure of the ZRANB3 HNH domain does not reveal the presence of a divalent cation in the putative active site, residues Asp1020, His1021 and His1045 assume equivalent positions to the catalytically important residues within the ββα-metal fold of T4 endonuclease VII (Fig. 3b). Sequence comparisons show strict conservation of Asp1020 and His1021 and His1045 among ZRANB3 proteins, highlighting their importance (Fig. 1c). By analogy with the T4 endonuclease VII structure, we hypothesized the involvement of Asp1020 and His1045 in metal coordination, and the role of His1021 as a general base in nucleophilic attack.

To address the relevance of specific residues in the ββα-fold of HNH domain, we introduced a number of mutations and tested the activities of the mutant ZRANB3 proteins. Although the mutations did not have a dramatic effect on DNA-dependent ATP hydrolysis (Supplementary Fig. 2c, Table 2), some of them either substantially reduced (N1036A), or completely abrogated (D1020A, H1021A and H1045A) ZRANB3 endonuclease activity (Fig. 3c). This is consistent with the proposed catalytic roles of Asp1020, His1021 and His1045 in the nucleolytic cleavage of DNA substrate.

ZRANB3 differs from other characterized HNH nucleases in being an ATP-dependent nuclease. Our previous work showed that mutation of the conserved Lys65 in the ATP-binding motif of ZRANB3 (known as the Walker A motif) yielded an ATPase-deficient enzyme9. Although the ATPase dead ZRANB3 mutant K65R showed no detectable endonuclease activity, it was unclear whether this was due to its inability to hydrolyse ATP, or due to the possible effect of the K65R mutation on the binding of ATP. To test whether ATP hydrolysis is essential for endonuclease activity, we tested ZRANB3 endonuclease activity in the presence of non-hydrolysable ATP analogues (ATPγS, AMP-PNP, AMP-PCP). Our data show that substitution of ATP by non-hydrolysable ATP analogues in ZRANB3 endonuclease reactions resulted in a complete loss of activity (Fig. 3d). ZRANB3 endonuclease activity is therefore strictly dependent on ATP hydrolysis catalysed by the helicase core domain.

Stimulation of nuclease activity by PCNA

Given that ZRANB3 acts as a structure-specific endonuclease, we compared its activity to an archetypal structure-specific endonuclease: FEN1. FEN1 is known to interact with PCNA through the PIP box located at its C-terminus5,6, and this association was shown to stimulate endonuclease activity of FEN1 (refs 23, 24). We therefore sought to test whether PCNA also has an effect on the endonuclease activity of ZRANB3. Our results show that, similarly to FEN1 activation, increasing concentrations of PCNA stimulated endonuclease activity of ZRANB3 (Fig. 4a). To gain molecular insight into this activation, we decided to explore the way ZRANB3 interacts with PCNA. Previous studies identified two conserved PCNA binding motifs in ZRANB3: a PIP box located between residues 519 and 526, and an APIM motif located at the very C-terminal end of the ZRANB3 protein (Fig. 4b, ref. 10). We inactivated the PIP box and the APIM motif by mutating critical residues (Fig. 4b,c), and assessed the impact of PCNA interactions on ZRANB3 endonuclease activity. Whereas the wild-type ZRANB3 showed notable stimulation of endonuclease activity in the presence of PCNA, such stimulation could not be observed upon inactivation of either the PIP box or the APIM motif (mutants PIP* and ΔAPIM, respectively) (Fig. 4d,e). An additive effect could not be observed with the PIP*-ΔAPIM double mutant, which highlights the relevance of both PCNA binding motifs for optimal ZRANB3 endonucleolytic activity. Interestingly, PCNA did not stimulate the ATPase activity of ZRANB3 (Supplementary Fig. 2d). Moreover, mutations of the PCNA binding motifs did not affect ATPase function (Supplementary Fig. 2e, Table 2), suggesting that ZRANB3 employs distinct mechanisms for activation (by ATP hydrolysis) and stimulation (by PCNA binding) of its endonuclease activity.

PCNA-dependent recruitment of ZRANB3

We next wanted to address the importance of PCNA in targeting structure-specific nucleases to sites of DNA damage. We expressed YFP-tagged proteins in U2OS cells and examined their ability to localize at sites of DNA damage caused by Ultraviolet laser microirradiation. As shown in Fig. 5a, YFP-FEN1 was efficiently recruited to the sites of laser-induced DNA damage. However, mutation of the PIP box motif resulted in a dramatic loss of FEN1 recruitment to the microirradiated stripes (YFP-FEN1 PIP* mutant), highlighting the importance of PCNA in mobilizing FEN1 to the appropriate nuclear locations. We further examined the recruitment of ZRANB3 to DNA damage, which was previously suggested to employ a similar PCNA-dependent mechanism9,10. To address the relevance of the individual PCNA binding motifs in ZRANB3 more directly, we expressed them as YFP-tagged proteins with nuclear localization signals. As shown in Fig. 5b, both the PIP box and the APIM motif were efficiently mobilized to the sites of DNA damage. However, this was completely abrogated when mutations, shown to abolish interactions with PCNA (Fig. 4c), were introduced into the PIP box and the APIM motif (Fig. 5b, mutants PIP*and APIM*). To address the relevance of these motifs in the context of the full length ZRANB3 protein, we further tested the recruitment of YFP-ZRANB3 wild-type and mutant proteins to locally induced DNA damage. In agreement with the published data10, ZRANB3 was readily recruited to the microirradiated stripes, and the individual inactivation of either the PIP box or the APIM motif (mutants PIP* and ΔAPIM, respectively) was not sufficient to abrogate recruitment to DNA damage (Fig. 5c). The recruitment of ZRANB3 was abolished only upon inactivation of both PCNA binding motifs (PIP*-ΔAPIM double mutant, Fig. 5c), indicating that the PIP box and the APIM motif might act independently in a biological context to support PCNA-dependent recruitment to DNA damage.

Figure 5: PCNA-dependent recruitment of ZRANB3. (a) Recruitment of FEN1 to the sites of laser induced DNA damage is abrogated by the mutations in the PIP box (Q337A, F343A and F344A). U2OS cells were transiently transfected with the indicated YFP constructs and analysed by live-cell imaging. Shown are representative images at the indicated time points post damage. (b) Recruitment of the YFP-tagged PIP box and APIM motif to the sites of laser induced DNA damage. Recruitment is abrogated by the mutations of the conserved residues in the PIP box and the APIM motif. (c) Recruitment of the YFP-tagged wild-type ZRANB3 and ZRANB3 PCNA binding mutants ZRANB3-PIP*, ZRANB3-ΔAPIM and ZRANB3-PIP*ΔAPIM) to the sites of laser induced DNA damage. (d) Recruitment of the wild-type and mutant ZRANB3 proteins to the sites of ongoing DNA replication. U2OS cells were transiently transfected with the indicated YFP constructs and stained against endogenous PCNA. Scale bar (a–d) 5 μm. (e) ZRANB3 accumulates at stalled replication forks. U2OS cells were transfected with YFP-ZRANB3 constructs and either left untreated, or exposed to UV irradiation. After 6 h, cells were fixed and stained with PCNA antibody. The percentage of cells containing ZRANB3 foci that colocalize with PCNA was determined. Shown is the average of three experiments. s.d.’s are shown as error bars. Full size image

We also examined the relevance of PCNA binding motifs in targeting ZRANB3 to the sites of DNA replication. To do this, we expressed YFP-ZRANB3 wild-type and mutant proteins and analysed their ability to colocalize with endogenous PCNA. In undamaged conditions, the majority of YFP-ZRANB3 expressing cells show uniform nuclear expression (such as in Fig. 5c). However, about 20% of the cells expressing YFP-ZRANB3 form foci, which colocalize with PCNA (Fig. 5d,e and Supplementary Fig. 4) (ref. 9). Individual mutation of either of the PCNA binding motifs in ZRANB3 reduced the efficiency of foci formation (PIP* and ΔAPIM mutants, Fig. 5e), but a complete loss of colocalization with PCNA was observed only upon inactivation of both motifs (PIP*-ΔAPIM double mutant, Fig. 5d,e). This indicates that the PIP box and the APIM motif mediated interactions with PCNA play critical roles in the recruitment of ZRANB3 to the sites of ongoing DNA replication.

ZRANB3 is also known to play a role in the replication stress response and to accumulate at sites of stalled DNA replication9,10,11. To address the significance of PCNA-binding motifs in recruiting ZRANB3 to stalled replication forks, we expressed YFP-ZRANB3 wild-type and mutant proteins in U2OS cells, and induced replication stress by exposing the cells to ultraviolet light (UV) irradiation. Following a 6-h recovery, we evaluated recruitment of ZRANB3 to stressed replication forks by measuring colocalization of YFP-ZRANB3 proteins with endogenous PCNA. UV induced an increase in the number of cells that formed ZRANB3 foci, and for wild-type ZRANB3 the proportion changed from ∼20% of cells in undamaged conditions to >95% following Ultraviolet irradiation (Fig. 5e and Supplementary Fig. 5). The number of cells containing focally concentrated ZRANB3 also increased with the PIP* and ΔAPIM mutants following Ultraviolet irradiation, but not with the PIP*-ΔAPIM double mutant, whose ability to form foci was completely lost (Fig. 5e). Interestingly, the increase in the number of cells containing ZRANB3 foci was more pronounced with the PIP* mutant than with the ΔAPIM mutant, possibly suggesting a specific role of the APIM motif in the replication stress response.

Comparative analysis of PCNA binding affinities

We further examined the association of PCNA and the peptides derived from ZRANB3 by performing isothermal titration calorimetry (ITC) (Supplementary Fig. 6). Analyses of the synthetic peptides containing the PIP box or APIM motif sequences revealed comparable affinities of the two peptides for PCNA (K D values of 4.8 and 9.24 μM for the PIP box and the APIM motif, respectively) (Supplementary Table 1). In agreement with previous studies, the association of the peptides with PCNA fitted a 1:1 stoichiometry, suggesting that the trimeric complex can accommodate one peptide per monomer5,25,26,27. Furthermore, the associations of ZRANB3 peptides were compared to the interactions of peptides derived from other PCNA interacting proteins (FEN1, p21 and Polι). The peptides were of equal length and covered the homologous sequence within the PIP box region. Our data showed that the affinities of ZRANB3 peptides were within the range of values detected for other PIP box peptides (Supplementary Table 1).

Structures of the PCNA-PIP and PCNA-APIM complexes

To gain molecular insight into the way the PIP box and the APIM motif of ZRANB3 interact with PCNA, we crystallized and determined the co-structures of PCNA with the ZRANB3 PIP and APIM peptides (Table 1). Both the PCNA:ZRANB3(PIP) and PCNA:ZRANB3(APIM) complexes show the typical homo-trimeric PCNA structure, consisting of three PCNA molecules in the asymmetric unit forming a trimeric ring28. Three peptide molecules are visible in the structures, each of which interacts with a different monomer in the PCNA trimer.

The PIP boxes found in PCNA interaction partners can generally be categorized as either canonical (defined by the Q-x-x-[ILM]-x-x-F-[FY] consensus sequence) or non-canonical (deviate from the canonical consensus). ZRANB3 has a canonical PIP box (Fig. 4b) that binds to the PCNA ring in a manner similar to other canonical PIP boxes (such as those found in p66 of Polδ and FEN1; Figs 6a–f and 7i)5. Specifically, the conserved glutamine (Gln519) in the PIP box peptide interacts with PCNA by forming a hydrogen bond with the main-chain carbonyl of Ala252, and by an additional contact with Ala208 via a well-structured water molecule (Fig. 6e,f). Furthermore, Lys518 and His520 form main chain hydrogen bonds with Ile255 and Pro253 of PCNA, respectively (Fig. 6e,f and Supplementary Fig. 8a). On the other hand, residues Ile522-Phe526 form a 3 10 helix that orients the conserved hydrophobic residues (also known as the ‘hydrophobic plug’1; Ile522, Phe525 and Phe526 in the ZRANB3 PIP box) for docking into the hydrophobic patch on the PCNA surface (Fig. 6d). While this topology is conserved in other PIP box peptides, differences are usually observed in the regions at the C-terminus of the PIP box sequence. This region forms an antiparallel β-sheet with the IDCL of PCNA in p21 (ref. 25) and FEN1 (ref. 6), but not in some other proteins with canonical PIP boxes, such as the PIP box in the p66 subunit of Polδ5 or the PIP box in the intrinsically disordered protein p15(PAF)29 (Fig. 7i). In the structure presented here, the PIP box peptide is not long enough to form extensive contacts with the IDCL.

Figure 6: Structure of the PCNA:ZRANB3(PIP) complex. (a) Front view of the PCNA ring (grey surface and ribbons) with the ZRANB3 PIP box peptide (yellow sticks). (b) Magnified view of the boxed region in a. Shown is a surface representation of one of the three PIP box binding sites on the PCNA ring with the bound PIP box peptide (yellow sticks coloured by atom type). (c) Overview of the hydrogen-bond interaction network between the ZRANB3 PIP box peptide (yellow) and PCNA (grey). Bound water molecule (purple sphere) and hydrogen bonds (yellow dotted lines) are shown. (d) Hydrophobic pocket on PCNA surface (grey) with conserved residues that form ‘hydrophobic plug’(Ile 522, Phe525 and Phe526; shown as yellow sticks) in the ZRANB3 PIP box peptide. (e,f) Magnified view of the hydrogen-bond interaction network between the ZRANB3 PIP box peptide (yellow) and PCNA (grey, labels in italics). Bound water molecule (purple sphere) and hydrogen bonds (yellow dotted lines) are shown. Full size image

Figure 7: Structure of the PCNA:ZRANB3(APIM) complex. (a) Front view of the PCNA ring (grey surface and ribbons) with the ZRANB3 APIM motif peptide (blue sticks). (b) Magnified view of the boxed region in e. Shown is a surface representation of one of the three APIM motif binding sites on the PCNA ring with the bound APIM motif peptide (blue sticks coloured by atom type). (c) Overview of the hydrogen-bond interaction network between the ZRANB3 APIM motif peptide (blue) and PCNA (grey). Bound water molecule (purple sphere) and hydrogen bonds (yellow dotted lines) are shown. (d) Hydrophobic pocket on PCNA surface (grey) with the residues that form ‘hydrophobic plug’ in the APIM motif (Ile 1072, Phe1075 and Leu1076; shown as blue sticks). (e–g) Magnified view of the hydrogen-bond interaction network between the ZRANB3 APIM motif peptide (blue) and PCNA (grey, labels in italics). Bound water molecule (purple sphere) and hydrogen bonds (yellow dotted lines) are shown. (h) Alignment of the APIM motifs from different human proteins. (i) Superimposed backbones of bound PIP box and APIM motif peptides from different co-crystal structures. Alignment of the peptides is shown on the right. Shown are ZRANB3-PIP515–529 (yellow; PBD:5MLO), FEN1336–348 (orange; PDB:1U7B), p21143–160 (red; PDB: 1AXC), Polη694–713 (magenta; PDB: 2ZVK), p1551–71 (cyan; PDB: 4D2G) and ZRANB3-APIM1065–1079 (blue; PDB:5MLW). Full size image

Although the APIM motif has been characterized as a PCNA-binding motif in a number of proteins3 (Fig. 7h), the structural insight into its mode of interaction with PCNA is still lacking. Our structure of PCNA:ZRANB3(APIM) shows that the APIM motif occupies the same general binding site on the PCNA surface as do canonical and non-canonical PIP boxes (Fig. 7a–c,i and Supplementary Fig. 8c)30. Moreover, the basic topology of the PIP box defined by a 3 10 helix is conserved in the APIM motif (Fig. 7d), despite the apparent differences in the amino-acid sequence. The 3 10 helical conformation is stabilized by a network of intramolecular hydrogen bonds; however, whereas in PIP boxes this network does not seem to necessitate conservation of the constituent residues, in the APIM motif it involves a conserved basic residue (Arg1074 in ZRANB3; Fig. 7h), whose side chain nitrogen interacts with the backbone carbonyl of Asp1071. Moreover, Ile1072, Phe1075 and Leu1076 of the APIM motif form a hydrophobic plug, analogously to the conserved hydrophobic residues of the PIP box [ILM]-x-x-F-[FY] sequence (Fig. 7d,i). As in the PIP box structure, the 3 10 helical configuration appropriately positions these residues into the hydrophobic patch on the PCNA surface.

Despite these similarities, the APIM motif interacts with PCNA in a specific way, which differs from previously characterized interactions of PCNA-interacting peptides. The most notable difference is the absence of a glutamine residue otherwise conserved in canonical PIP boxes (Fig. 7i), whose side chain forms both direct and water mediated hydrogen bonds with PCNA25,29 (Fig. 6e). The APIM motif contains a glycine at the analogous position (Gly1069), and is therefore unable to interact with PCNA in the same manner; instead, it forms a main chain–main chain hydrogen bond with Ile255 (Fig. 7f and Supplementary Fig. 8b). In addition, the side chain hydroxyl group of Ser1070 forms water mediated hydrogen bonds with Ala252 and Ala208, while Ile1072 forms a main chain hydrogen bond with His44 (Fig. 7e and Supplementary Fig. 8b). Differences are also observed in the formation of hydrophobic interactions: Leu1076 of the tripartite hydrophobic plug in the APIM motif has a smaller side chain than the corresponding phenylalanine of the PIP box peptide, and does not intrude as deeply into the PCNA binding pocket. This is not dissimilar to the non-canonical PIP box interaction of Polι with PCNA, which also contains a leucine residue at the corresponding position31.

The differences in the way the PIP box and the APIM motif of ZRANB3 bind to PCNA extend beyond the hydrophobic pocket on the PCNA surface. The C-terminal region of the APIM motif interacts with the IDCL, providing an additional point of contact. Specifically, the Val1077 of the APIM motif forms main-chain hydrogen bonds with Gly127 of PCNA (Fig. 7g). Interestingly, the corresponding region of Polη interacts with PCNA in a similar fashion and binds the same IDCL residue through equivalently positioned Lys709 (ref. 31). This differs from the extensive antiparallel β-sheets observed in the p21 and FEN1 PCNA complexes, which involve multiple intramolecular contacts25,29.

Biological implications of ZRANB3 structural studies

Replication stress is one of the major factors that contributes to genomic instability associated with the development of human cancers32,33,34. Although the role of ZRANB3 in the replication stress response has been evidenced by a number of studies9,10,11,35, pathobiological importance of its function has not been investigated. Interestingly, multiple ZRANB3 variants have been identified in various human cancers, including endometrial carcinomas, a malignancy characterized by marked genomic instability36,37. Mapping the endometrial cancer associated ZRANB3 variants onto the primary sequence of ZRANB3, we noted that the majority localized to functionally relevant domains, particularly the helicase core and the HNH domain38,39 (Fig. 8a). Furthermore, bioinformatic analyses (largely based on evolutionary conservation of the affected amino acids and on structural properties) suggested high probabilities of pathogenicity for majority of the ZRANB3 variants Supplementary Table 2.

Figure 8: Cancer associated ZRANB3 mutations. (a) ZRANB3 mutations associated with endometrial carcinomas. Shown are positions of the mutations targeting conserved residues. Truncating mutations are indicated as black dots. (b) Nuclease assay with the wild-type and cancer associated mutant ZRANB3 proteins, in the presence and absence of ATP. Reactions were analysed by native polyacrylamide gel electrophoresis. Indicated are mobilities of the fluorescently labelled DNA substrate and product. (c) Quantification of the ATPase and nuclease activities of the wild-type and mutant ZRANB3 proteins. Shown are the averages of three ATPase and five nuclease reactions. s.d.’s are shown as error bars. Full size image

Three of the variants are predicted to yield truncated products (nonsense mutations E97*, R947* and a frameshift mutation C1041Hfs*13, Supplementary Table 3). Among these, E97* is expected to produce a functionally null allele, whereas R947* and C1041Hfs*13 are both expected to yield endonuclease deficient ZRANB3. Intriguingly, we noted an elevated frequency of highly conserved ZRANB3 residues among the mutations reported in the study (T66A, K340T, G401D, F414C and D1020Y), among which T66A and D1020Y are predicted to yield enzymatically inactive ZRANB3. Specifically, Thr66 is a conserved residue of a common nucleotide binding fold (known as the Walker A motif, defined by the G-X(4)-G-K-[TS] sequence), and its mutation is expected to yield both ATPase and endonuclease deficient ZRANB3. On the other hand, Asp1020 was identified in this study as an active site residue of the HNH endonuclease domain (Fig. 3c), and its mutation to tyrosine could be predicted to ablate the ZRANB3 endonuclease activity.

To investigate the functional significance of cancer associated ZRANB3 mutations, we purified relevant ZRANB3 mutant proteins and tested their enzymatic activities. The majority of the tested cancer associated mutations yielded nucleolytically inactive ZRANB3 proteins (Fig. 8b,c). While in some mutants, such as R947Q, R947* and D1020Y, this was not related to the ATPase function (Fig. 8c, Table 2), in others the mutations caused ablation of both ATPase and endonuclease activities. Some mutations (K340T, F414C and C1041Hfs*13) resulted in insoluble proteins.

Collectively, these data suggest a high frequency of loss-of-function mutations in ZRANB3 that may impact on its role in the replication stress response, and consequently on genomic stability.