Structural insights into Rad18 targeting by the SLF1 BRCT domains

Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18 recognition by SLF1 is unclear. Here, we present the crystal structure of the tandem BRCT repeat (tBRCT) in SLF1 (SLF1tBRCT) bound with the interacting Rad18 peptide. Our structure and biochemical studies demonstrate that SLF1tBRCT interacts with two phosphoserines and adjacent residues in Rad18 for high-affinity and specificity Rad18 recognition. We found that SLF1tBRCT utilizes mechanisms common among tBRCTs as well as unique ones for Rad18 binding, the latter include interactions with an α-helical structure in Rad18 that has not been observed in other tBRCT-bound ligand proteins. Our work provides structural insights into Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated complex assembly.

Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair.The mechanism of the specific Rad18 recognition by SLF1 is unclear.Here, we present the crystal structure of the tandem BRCT repeat (tBRCT) in SLF1 (SLF1 tBRCT ) bound with the interacting Rad18 peptide.Our structure and biochemical studies demonstrate that SLF1 tBRCT interacts with two phosphoserines and adjacent residues in Rad18 for high-affinity and specificity Rad18 recognition.We found that SLF1 tBRCT utilizes mechanisms common among tBRCTs as well as unique ones for Rad18 binding, the latter include interactions with an α-helical structure in Rad18 that has not been observed in other tBRCT-bound ligand proteins.Our work provides structural insights into Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated complex assembly.
The conserved Rad18 protein plays multiple roles in maintaining genome stability.Its best-known function is to monoubiquitinate the proliferating cell nuclear antigen (PCNA), which signals for translesion DNA synthesis and enables PCNA poly-ubiquitination for additional DNA damage tolerance responses (1)(2)(3)(4).Recently, Rad18 has been shown to interact with the structural maintenance of chromosome (SMC)5/6 localization factor 1 (SLF1) (5,6).The SMC5/6 complex has important functions in DNA repair (7,8).It was found that through interacting with SLF1, Rad18 recruits the SMC5/6 complex to DNA damage sites for repair (9,10).The importance of the Rad18-SLF1 pathway is highlighted by the fact that its deficiencies are associated with chromosomal instability and developmental defects (11).
Previous studies have shown that the tandem BRCA1 C-Terminal (BRCT) domain repeat (tBRCT) at the SLF1 N-terminus (SLF1 tBRCT ) plays a critical role in the Rad18-SLF1 association (5,6).BRCT domains were first identified in the tumor suppressor BRCA1 protein and subsequently found in more than 20 human proteins, most are involved in genome maintenance (12,13).In most cases, BRCT domains are assembled as tBRCT units, though units with single or more than two BRCT domains have also been found.Many tBRCTs specifically interact with phosphorylated ligand proteins, which often arise during DNA replication, DNA damage repair, and checkpoint response.Such phosphorylationdependent protein-protein interaction plays key structural roles in the assembly of protein complexes in these processes as well as in the recruitment of proteins to specific genomic sites (14)(15)(16)(17).
Structural studies revealed that the specificity of tBRCTmediated ligand recognition is determined by both a phosphorylated serine or threonine residue and adjacent residues in the ligand proteins.The phosphorylation of Ser442 and Ser444 in Rad18 is critical for the Rad18-SLF1 association (5).However, the adjacent Rad18 region shares no obvious sequence similarities with previously characterized tBRCT ligand proteins (Table S1).The molecular basis for the specific Rad18 targeting by SLF1 has been unclear.
Here, we demonstrate that SLF1 tBRCT interacts strongly and specifically with the Ser442/Ser444-containing Rad18 peptide upon phosphorylation of these serine residues.We determined the crystal structure of SLF1 tBRCT with bound Rad18 peptide.Structure-guided functional studies revealed that SLF1 tBRCT interacts with both phosphoserines as well as adjacent regions.Similar to other tBRCTs, two pockets in SLF1 tBRCT recognize phosphoserine 442 and a neighboring residue in Rad18.In addition, SLF1 tBRCT also forms three sets of interactions with Rad18 that diverge from interactions observed in other tBRCT-ligand complexes.Our study provides structural insights into the high affinity and high specificity Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated protein-protein interactions.
To assess the specificity of the SLF1 tBRCT -Rad18 interaction, we probed the interaction of SLF1 tBRCT with a phosphopeptide optimized for binding to the BRCA1 tBRCT (BRCTide, GAAYDI(pS)QVFPFAKKK) (18).ITC experiments indicated that SLF1 tBRCT interacted weakly with BRCTide, with a K D of 27.5 μM that is 2000-fold higher than the K D for the Rad18-2P peptide (Fig. 1B and Table 1).Collectively, our data suggest that Ser442 and Ser444 phosphorylation enables strong and specific interaction between the Rad18 Ser442/Ser444containing region and SLF1 tBRCT .

Structure of SLF1 tBRCT in complex with a dually phosphorylated Rad18 peptide
The strong and specific Rad18-2P-SLF1 tBRCT interaction enabled us to crystalize their complex and determine the structure to a resolution of 1.75 Å (Table 2).SLF1 residues 5 to 199 are resolved in the electron density map, which forms a compact and elongated structure with a dimension of 35 x 35 x 65 Å (Fig. 1C).The two SLF1 BRCT domains (BRCT 1 and 2) adopt a typical BRCT fold, consisting of a central 4-strand parallel β-sheet flanked by two α-helices on one side and one on the other.They are arranged in parallel orientation and form primarily hydrophobic interactions with each other.The SLF1 tBRCT structure is further stabilized by the three-helix linker that interacts with both BRCT domains.Two SLF1 tBRCT molecules were found in the asymmetric unit.Most regions in them adopt very similar structures and can be aligned with a root mean square deviation (RMSD) of 0.69 Å for the Cα atoms (Fig. S2A).Structural differences were observed for part of the β1B-α1B loop (letters A and B indicate the first and second BRCT domains, respectively) and the α2Bspanning region, suggesting the structural flexibility of these regions.Both regions participate in crystal packing interactions, which may stabilize the observed structure.
Strong densities for the Rad18-2P peptide were found on both SLF1 tBRCT molecules in the crystal (Figs.1D and S2B).Rad18 residues 437 to 452 and 436 to 451 were fitted into these densities on SLF1 tBRCT molecules 1 and 2, respectively, revealing mostly identical structures for the two Rad18-2P peptides (Fig. S2A).The Rad18 binding site in SLF1 tBRCT is composed of both BRCT domains and predominantly positively charged (Fig. S2C).The Rad18 peptide N-terminal half including the phosphoserines adopts an extended conformation and interacts with BRCT 1, its C-terminal half forms an α-helix and interacts primarily with BRCT 2 (Fig. 1C).The Rad18-2P-SLF1 tBRCT interface is the largest among tBRCT-ligand peptide structures reported to date, burying 1600 Å 2 of surface area.
Phosphoserine 442 and 444 in Rad18 make distinct contributions to SLF1 binding Our structure revealed that phosphoserines 442 and 444 (pS442 and pS444) in Rad18 form distinct interactions with SLF1 tBRCT (Figs.2A, S2D, and S3, A and B).The pS442 side chain is completely buried at the Rad18-SLF1 interface.Its phosphate group forms polar interactions with the SLF1 Thr13 and Lys56 side chains and the Gly14 main chain carbonyl.In contrast, the pS444 side chain is mostly exposed to solvent.Its phosphate group forms polar interactions with the SLF1 Arg50 side chain in complex 2 in the crystal (Fig. S2D).It also interacts with the Arg448 side chain in Rad18 (Figs. 2A and  S2D).
To probe the function of pS442 and pS444 in the Rad18-SLF1 tBRCT association, we evaluated the effects of removing one of the phosphate groups or substituting SLF1 residues they interact with.ITC experiments revealed that removing the pS442 phosphate group or alanine substitution of its interacting residue Lys56 in SLF1 tBRCT strongly inhibited the Rad18-2P-SLF1 tBRCT interaction, resulting in 1300-and 150fold increases in K D , respectively (Fig. 2, B and C and Table 1).In contrast, moderate effects were observed for removing the pS444 phosphate group or alanine substitution of its interacting residue Arg50.Both modifications caused a 10-fold increase in K D (Fig. 2, B and C and Table 1).The gel filtration elution profiles for the R50A-and K56A-substituted SLF1 tBRCT proteins are very similar to the unsubstituted protein (Fig. S4), suggesting that the reduced affinity exhibited by these substituted proteins are not due to changes in the overall SLF1 tBRCT structure but loss of specific interactions at the SLF1 tBRCT -Rad18 interface.Together these data suggest that Ser442 phosphorylation is critical for the Rad18-SLF1 tBRCT association, whereas Ser444 phosphorylation plays a less important role and enhances the binding affinity.
To validate the ITC data, we carried out in vitro pull-down experiments with purified full-length Rad18 and SLF1 tBRCT .Serine-to-glutamate substitutions were introduced into Rad18 to mimic the Serine 442 and/or 444 phosphorylation.The pulldown experiments indicated that SLF1 tBRCT strongly coprecipitated with the S442E-or S442E/S444E (2SE)-substituted Rad18, but not the wild type or the S444E-substituted Rad18 (Fig. 2D).These pull-down data are in line with the notion that Ser442 and Ser444 phosphorylation make different contributions to the Rad18-SLF1 tBRCT association.However, these data do not exclude the role of pS444 in promoting Rad18-SLF1 tBRCT association, as the serine-to-glutamate substitution does not fully replicate the physiological functions of serine phosphorylation.Structure of SLF1 tBRCT in complex with a Rad18-pS442 peptide Our binding experiments suggest that Ser442 phosphorylation is critical and sufficient for strong Rad18-SLF1 tBRCT association.To further clarify the roles of Ser442 and 444 phosphorylation in the Rad18-SLF1 interaction, we cocrystallized SLF1 tBRCT with the singularly phosphorylated Rad18-pS442 peptide and determined its structure to a resolution of 1.62 Å (Table 2).Two SLF1 tBRCT molecules were found in the asymmetric unit and electron densities for the Rad18-pSer442 peptide were found on both of them (Figs.3A  and S5A).The structure and crystal packing of the SLF1 tBRCT -Rad18-pS442 complex are very similar to the SLF1 tBRCT -Rad18-2P complex (Fig. 3B).Compared to the SLF1 tBRCT -Rad18-2P complex, little structural differences were observed even for the SLF1 Arg50 and Rad18 Arg448 side chains, which interact with the pS444 phosphate group in the Rad18-2P peptide (Figs.3C  and S5B).The structure is consistent with the critical role of Ser442 phosphorylation in the Rad18-SLF1 association.
The electron densities for the Rad18-pS442 peptide are weaker compared to electron densities for the Rad18-2P peptide, especially at the N-terminal region where Ser442 and Ser444 reside (Figs.3A and S5A).Such a difference in density is in line with the reduced affinity of the Rad18-pS442 peptide toward SLF1 tBRCT and supports the function of Ser444 phosphorylation in enhancing the Rad18-SLF1 tBRCT binding.
Electron densities for the Arg448 side chain in the Rad18-pS442 peptide are also weaker, consistent with the lack of Arg448-pS444 interaction in this peptide.
Rad18 regions adjacent to Ser442 and Ser444 are critical for interaction with SLF1 tBRCT Our structures revealed that Rad18 residues adjacent to Ser442 and Ser444 form two additional interfaces with SLF1 tBRCT .The Rad18-2P peptide N-terminal region forms several hydrogen bonds with SLF1 tBRCT .Its Cys439 main chain carboxyl hydrogen bonds with the SLF1 Lys36 side chain, Asn440 side-chain hydrogen bonds with the SLF1 Lys20 side chain, and the Phe15 main chain amide (Figs.4A, S3, A and B, and S6A).In complex 1 in the crystal, its Ser441 side chain hydrogen bonds with the SLF1 Glu38 side chain (Fig. 4A), whereas in complex 2 the SLF1 Glu38 side chain interacts with the Ser441 main chain amide through a water molecule (Fig. S6A).Cys439 in this region also forms hydrophobic interactions with Met17 in SLF1.The C terminal half of the Rad18 peptide adopts an α-helical structure, presenting residues Ile446, Ile447, Leu450 and Leu451 for interactions with a large hydrophobic surface in SLF1 consist of residues Leu58, Val133, Ser141, Leu142, Val145, Ile189, Leu192 and Gly193 (Figs. 4B, S3, A and B, and S6B).The α-helix is stabilized by the pS444-Arg448 interaction and a Ser443-Asp449 hydrogen bond.The N-terminus of the helix and neighboring region pack against the SLF1 52-55 region, with the pS444 carbonyl and Asp445 amide groups in this region forming hydrogen bonds with the mainchain amide and carbonyl groups of Lys53 in SLF1, respectively.The same regions in the Rad18-pS442 peptide form virtually identical interactions with SLF1 tBRCT , Figure 3. Structure of the SLF1 tBRCT -Rad18-pS442 complex.A, electron density for the Rad18-pS442 peptide.The density for the Rad18 peptide bound to SLF1 tBRCT molecule 1 in the crystal contoured at 1σ is presented.B, comparison of the asymmetric unit structures.Superimposition of asymmetric unit structures of the SLF1 tBRCT -Rad18-pS442 complex and the SLF1 tBRCT -Rad18-2P complex (gray) is shown.The RMSD for the equivalent Cα atoms is 0.26 Å. C, interactions mediated by pS442 and surrounding regions in the Rad18-pS442 peptide.Structure of complex 1 in the crystal is presented.although the pS444-Arg448 interaction is absent in this peptide.
To probe the function of the above-mentioned SLF1-Rad18 interfaces, we introduced substitutions into the corresponding Rad18 regions and evaluated their effects on the Rad18-SLF1 tBRCT interaction.ITC experiments revealed moderate inhibitions of interaction for the N440A substitution at the Rad18 peptide N-terminus and the L450A/L451A (2LA) substitution at the C-terminal half of the Rad18 α-helix, which caused 3-fold increases in K D (Fig. 4, C and D and Table 1).In contrast, strong inhibitions were observed for the I446A/ I447A (2IA) substitution at the N-terminal half of the Rad18 αhelix and its combination with 2LA (4A).Both substitutions reduced the Rad18-SLF1 tBRCT interaction to undetectable levels (Fig. 4D).To further probe the function of the Rad18 αhelix in binding SLF1 tBRCT , we introduced the R448A and D449A substitutions in Rad18 to disrupt interactions stabilizing the α-helix, and alanine substitutions of Leu142 and Ile189 in SLF1 tRBRCT that interact with it.ITC experiments revealed that these substitutions increased the K D 2-to 8-fold (Fig. 4, C and E and Table 1).Gel filtration experiments suggest that the L142A and I189A substitutions do not alter the overall SLF1 tBRCT structure (Fig. S4).Together, these data support the important role of the Rad18 α-helix in its interaction with SLF1 tBRCT .Interestingly, the L142A substitution in SLF1 tBRCT caused a much stronger inhibition than the I189A substitution.Leu142 and Ile189 interact primarily with the Nand C-terminal halves of the Rad18 α-helix, respectively.Such data are in line with the notion that the N-terminal half of the Rad18 α-helix plays a more important role than the C-terminal half in interacting with SLF1 tBRCT .
Together, our structure and the ITC data suggest that Rad18 regions adjacent to Ser442 and Ser444 also contribute to the Rad18-SLF1 tBRCT interaction, among which the α-helix in Rad18 plays a particularly important role.
The previously characterized tBRCTs contain two common ligand-binding pockets.The first located in BRCT 1 is conserved and interacts with the anchoring phosphoserine or phosphothreonine in the ligand peptide, the second is located between the two BRCT domains and recognizes the +3 ligand residue (3 residues C-terminal to the anchoring residue) (16,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33).Equivalent pockets can be found in SLF1 tBRCT .The first interacts with pS442 in Rad18 and the interactions are similar to the anchoring residue-mediated interactions in other tBRCT-ligand complexes (Fig. 5B).Residues constituting this binding pocket, Thr13, Gly14, and Lys56, are conserved among tBRCTs (Fig. 5A).The second binding pocket recognizes Ile446 in Rad18.It is relatively deep and structurally similar to the second pocket in Brc1, Rtt107, and TopBP1 (Fig. 5C).Ser141 in SLF1 or the equivalent residues in Brc1, Rtt107, and TopBP1 located at the bottom of the pocket contain small side chains, leading to the formation of a deep pocket.Additional tBRCTs contain a shallow second ligand binding pocket, either due to the large side chains of Leu1887 and H720/H723 in 53BP1 and Crb2, respectively; or the different β1B-α1B loop conformation in BRCA1, MDC1, PTIP and MCPH1 (Fig. 5D).These observations suggest that tBRCTs utilize several common mechanisms for ligand recognition.
The structural comparison revealed three sets of interactions at the Rad18-SLF1 tBRCT interface not previously observed in other tBRCT-ligand complexes.The first involves interactions with Rad18 residues N-terminal to Ser442 (Fig. 4A).Although several other tBRCT ligand peptides contain multiple residues N-terminal to the anchoring residue (Table S1), they adopt different structures and do not form similar interactions with tBRCTs.The second is the polar interaction between pS444 in Rad18 and Arg50 in SLF1 tBRCT .Though some other tBRCTs can bind dually phosphorylated ligand peptides (32,34), their interactions with the nonanchoring phosphorylated ligand residue do not resemble the pS444-Arg50 interaction between Rad18 and SLF1.Rather the latter bears partial similarity to the interaction between Arg704 in Brc1 and Glu +2 in the bound H2A tail (Fig. 5E).Equivalent residues in other tBRCT-ligand pairs are located too far apart to form a similar interaction.The third is the hydrophobic interactions mediated by the α-helix in Rad18 and a cleft between α1B, α3B, and the β1B-α1B loop in SLF1 tBRCT .Unlike the Rad18 peptide, all the previously characterized tBRCT-bound ligand peptides adopt extended conformations.In most of the other tBRCTs, the different conformations of the β1B-α1B loop and neighboring regions lead to the formation of a smaller cleft incompatible with α-helix binding (Fig. 5F).In Crb2, the polar side chains of His720 and His723 make the cleft unfavorable for hydrophobic interactions (Fig. 5D).The unique structure of Rad18 presents its Ile446 at the +4 position relative to pS442 for interaction with the second ligand binding pocket in SLF1 tBRCT .In contrast, the same pocket in other tBRCTs binds the +3 ligand residue.Collectively, these unique interactions at the Rad18-SLF1 tBRCT interface contribute to the highly specific Rad18 targeting by SLF1 tBRCT .

Discussion
Our structural and biochemical analyses reveal the molecular basis for the Rad18-SLF1 tBRCT interaction.We found that

Structure of SLF1 tBRCT bound with Rad18 peptide
Ser442 phosphorylation and a unique α-helix in Rad18 are critical for the Rad18-SLF1 tBRCT association, in which Rad18 residues N-terminal to Ser442 and its Ser444 phosphorylation also play important roles.Such a quadripartite binding mechanism explains the high affinity and specificity binding between the phosphorylated Rad18 peptide and SLF1 tBRCT .Structural comparison revealed that while SLF1 tBRCT and other tBRCTs utilize two common pockets for ligand recognition, the Rad18-SLF1 tBRCT interface also contains additional interactions not found in other tBRCT-ligand complexes.Thus, specific targeting of Rad18 by SLF1 tBRCT entails both mechanisms common among tBRCTs and unique mechanisms.
Rad18 residues involved in its recognition by SLF1 tBRCT are conserved in higher vertebrates but not in fishes or amphibians (Figs.1A and S1A).In contrast, SLF1 orthologs in most vertebrates contain an N-terminal tBRCT (Fig. S1B).Therefore, the Rad18-SLF1 tBRCT interaction probably emerges late in evolution and SLF1 tBRCT may possess functions beyond Rad18 targeting.In yeast, the SLF1 homolog Nse5 does not contain the tBRCT domains.However, its partner Nse6 can interact with the tetra-BRCT domain in Rtt107/Brc1, which also contains a tBRCT domain that binds to γH2A at DNA lesion sites.Through interaction with Nse6, Rtt107/Brc1 recruits the SMC5/6 complex to DNA damage sites (17,(35)(36)(37).Thus, in both yeast and higher eukaryotes, BRCT domains can target the SMC5/6 complex to DNA lesion sites though the exact mechanism varies.
Previous cellular studies indicated that Rad18 interacts with SLF1 in a phosphorylation-dependent manner and the interaction is abolished by the combinatory S442A/S444A substitution in Rad18 (5,9).Therefore, the mechanism determined here is likely critical for the interaction between the full-length SLF1 and Rad18 in vivo.The kinases phosphorylating Ser442 and Ser444 in Rad18 are currently unknown.Future studies to identify these kinases will help to better understand the Rad18-SLF1 signaling cascade.The Rad18 pS444 side chain is solvent exposed in our structure, suggesting that it has the potential for binding additional factors to relay the phosphorylation signal.Future studies are required to test this idea and further define the pathways involving Rad18, SLF1, and the SMC5/6 complex.

Protein expression and purification
The coding region for SLF1 tBRCT (residues 1-199) was optimized for expression in E. coli, synthesized (Sangon Biotech), and inserted into the vector pET26b (Novagen).The recombinant protein contains a C-terminal 6× histidine (his-) tag.For protein expression, E. coli BL21 Rosetta (DE3) cells transformed with this plasmid were cultured in LB medium supplemented with 34 mg/l kanamycin and 25 mg/l chloramphenicol and induced with 0.25 mM Isopropyl β-D-1thiogalactopyranoside (Meilunbio) at 16 C for 16 h.Collected cells were lysed with an AH-2010 homogenizer (ATS Engineering) and SLF1 tBRCT was purified by nickelnitrilotriacetic acid agarose (Smart Life Sciences), heparin (Hitrap Heparin HP, GE Healthcare) and gel filtration (Superdex 75 Increase 10/300, GE Healthcare) columns.The protein was concentrated to 10 mg/ml in a buffer containing 20 mM Tris (pH 7.5), 200 mM sodium chloride and 2 mM dithiothreitol (DTT), flash-cooled in liquid nitrogen, and stored at −80 C.
The coding region for the human Rad18 was optimized for expression in E. coli, synthesized (Sangon Biotech) and inserted into the vector pET28A (Novagen).Strep-, his-and sumotags were introduced to the Rad18 N-terminus by a PCR-based protocol.The recombinant triple tagged Rad18 was expressed in E. coli BL21 Rosetta (DE3) cells and purified with streptactin (Smart Life sciences), heparin (Hitrap Heparin HP, GE healthcare) and gel filtration (Superdex 200 10/300, GE healthcare) columns.The protein was concentrated to 10 mg/ ml in a buffer containing 20 mM Tris (pH 7.5), 200 mM sodium chloride, and 2 mM DTT; flash-cooled in liquid nitrogen; and stored at −80 C. Amino acid substitutions were introduced with a PCR-based protocol and verified by DNA sequencing.The substituted proteins were expressed and purified following the same protocols for the wild-type proteins.

Crystallization and structure determination
Prior to crystallization experiments, the SLF1 tBRCT solution was supplemented with 5× molar excess of Rad18-2P or Rad18-pS442 peptides (Sangon Biotech, Hefei Scierbio-Tech).Crystallization experiments were performed with a sittingdrop setup at 18 C.The reservoir solution for crystals with the Rad18-2P peptide contains 0.19 M magnesium chloride, 0.1 M Bis-Tris (pH6.5), and 24% polyethylene glycol (PEG) 3350; for crystals with the Rad18-pS442 peptide contains 0.3 M magnesium chloride, 0.1 M Bis-Tris (pH6.5) and 24% PEG3350.Prior to diffraction experiments, crystals were equilibrated in the reservoir solution supplemented with 25% (v/v) 2-propanol and flashed cooled in liquid nitrogen.Diffraction data for crystals containing the Rad18-2P peptide was collected at the National Facility for Protein Science beamline BL19U1 at Shanghai Synchrotron Radiation Facility (SSRF) at 0.9785 Å and indexed, integrated, and scaled with the HKL3000 package (38).The structure was determined with molecular replacement with PHASER (39), with the structure of the TopBP1 BRCT7/8 repeat (PDB 3AL2) as the search model.Diffraction data for crystals containing the Rad18-pS442 peptide was collected at the SSRF beamline BL02U1 at 0.9792 Å and indexed, integrated, and scaled with the XDS package (40).The structure was determined with molecular replacement with PHASER, with the structure of SLF1 tBRCT as the search model.Inspection and modification of the structures were carried out with O (41) and COOT (42).The structures were refined with PHENIX (43).

Pull-down experiments
To characterize Rad18 binding to SLF1 tBRCT , 50 μg hisstrep-sumo-tagged Rad18 was incubated with 30 μl streptactin beads (Smart Life sciences) in a binding buffer containing 20 mM Tris (pH 7.5), 200 mM sodium chloride and 2 mM DTT for 60 min at 4 C.After washing with 800 μl binding buffer three times, the beads were incubated with 50 μg SLF1 tBRCT at 4 C for 60 min.The beads were washed again with 800 μl binding buffer three times and the bound proteins were eluted with the binding buffer supplemented with 10 mM D-desthiobiotin (Sigma) and analyzed with SDS PAGE.

Figure 1 .
Figure 1.Structure of the SLF1 tBRCT -Rad18-2P complex.A, sequence alignment of Rad18.Residue numbers and secondary structure elements for the human Rad18 are indicated.Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Rn, Rattus norvegicus (rat); Gg, Gallus gallus (chicken); Mamu, Mauremys mutica (turtle).B, ITC experiments probing the interaction between SLF1 tBRCT and Rad18 peptides or BRCTide.C, crystal structure of the SLF1 tBRCT -Rad18-2P complex.SLF1 BRCT domains 1, 2 and the linker are colored in green, blue and yellow, respectively.Rad18 is colored in magenta.This color scheme is used throughout the manuscript unless otherwise indicated.Secondary structure elements and the N-and C-terminus of SLF1 tBRCT and the Rad18 peptide are indicated.D, electron density for the Rad18-2P peptide.The density for the peptide bound to SLF1 tBRCT molecule 1 in the crystal contoured at 1σ is presented.Structure figures are prepared with pymol (www.pymol.org).

Figure 2 .
Figure 2. Phosphoserine 442 and 444 in Rad18 make distinct contributions to its interaction with SLF1 tBRCT .A, interactions mediated by pS442, pS444, and surrounding regions in Rad18.The structure of complex 1 in the crystal is presented.Important residue side chains are highlighted.Dashed lines indicate hydrogen bonds or polar interactions.B, ITC experiments probing the interaction between SLF1 tBRCT and Rad18 peptides.C, ITC experiments probing the interaction between the Rad18-2P peptide and substituted SLF1 tBRCT .Data for the wild-type SLF1 tBRCT is included for comparison.D, Rad18-SLF1 tBRCT pull-down experiments.Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis of SLF1 tBRCT co-precipitated with the wild type or substituted Rad18 is presented.In lane "Ctrl", SDS PAGE analysis of the experiment without Rad18 is presented.

Figure 4 .
Figure 4. Rad18 regions adjacent to Ser442 and Ser444 make important contributions to its interaction with SLF1 tBRCT .A and B, interactions mediated by the N-(A) and C-terminal (B) regions of the Rad18-2P peptide.Structure of complex 1 in the crystal is presented.C and D, ITC experiments probing the interaction between SLF1 tBRCT and substituted Rad18-2P (C) or Rad18-pS442 (D) peptides.Data for the wild-type Rad18 peptides are included for comparison.E, ITC experiments probing the interaction between the Rad18-2P peptide and substituted SLF1 tBRCT .Data for the wild-type SLF1 tBRCT is included for comparison.
ITC experiments were carried out on a MicroCal ITC 200 instrument (Malvern) at 25 C. Prior to the ITC experiments, SLF1 tBRCT was exchanged into a buffer containing 20 mM Tris (pH 7.5) and 200 mM sodium chloride and peptides (Sangon Biotech, Scilight biotechnology, Hefei Scierbio-Tech, Synpeptide) were dissolved in the same buffer.To characterize binding, a solution containing 1.3 mM Rad18 peptide or 6 mM BRCTide was injected into a 300-μl cell that stores 0.1 mM (for binding experiments with Rad18 peptides) or 0.2 mM (for the binding experiment with BRCTide) SLF1 tBRCT , 2 μl at a time.Data were analyzed with ORIGIN 7.0 (Originlab).Three independent repeats were performed for each ITC experiment, representative data are presented in Figures1, 2, and 4.

Table 1
Summary of ITC experiments