Multifaceted Recognition of Vertebrate Rev1 by Translesion Polymerases ζ and κ*

Background: Translesion synthesis requires the scaffolding function of the Rev1 CTD. Results: We determined the structures of the Rev1 CTD and its complex with Pol κ and mapped its Rev7-binding surface. Conclusion: Distinct surfaces of the Rev1 CTD separately mediate the assembly of extension and insertion translesion polymerase complexes. Significance: Cancer therapeutics could be developed by inhibiting Rev1 CTD-mediated translesion synthesis. Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

An alarming number of DNA lesions are generated continually in living cells from endogenous cellular processes and from exposure to exogenous genotoxic agents (1,2). DNA lesions that have escaped from sophisticated DNA repair mechanisms such as nucleotide and base excision repair cannot be utilized as a template by high-fidelity replicative DNA polymerases, resulting in blockage of the replication fork and formation of replication gaps. To promote cell survival and complete DNA replication before mitosis, cells employ specialized DNA polymerases to bypass lesions in a damage tolerance process known as translesion synthesis at the cost of replication fidelity (3,4).
Translesion synthesis in mammalian cells is carried out by four Y-family polymerases, Pol 3 , Pol , Pol , and Rev1, and one heterodimeric B-family polymerase Pol that consists of the Rev3 catalytic subunit and the Rev7 accessory subunit. Accumulating evidence suggests that translesion synthesis is often achieved in a two-step fashion, with one set of polymerases carrying out insertion opposite the lesion followed by a second set of polymerases carrying out the extension (5). The insertion step is achieved by one of the Y-family polymerases , , , or Rev1, with each specialized at bypassing distinct lesions (cognate lesions) for optimal replication accuracy (4). In addition to cognate lesions, these insertion polymerases can also bypass a variety of other lesions, often redundantly, though with elevated mutagenic rates. In contrast, many of the translesion synthesis events require the action of the Rev1-Pol complex for efficient primer extension regardless of lesion types (5). Accordingly, deletions of REV1 and genes encoding the subunits of Pol , REV3, and REV7, exhibit reduced spontaneous mutation rates and are severely defective for mutations induced by a wide variety of DNA-damaging agents (6 -8). Recently, translesion synthesis has also been shown to function in replication-coupled DNA interstrand cross-link repair in a Pol -dependent manner (9), and monoubiquitinated Rev1 has been found to interact with the Fanconi anemia core complex, providing a critical link between the Fanconi anemia pathway and translesion synthesis activity (10). These results have further expanded the function of translesion synthesis and highlight the fundamental roles of Rev1 and Pol in this biologically important process.
The overlapping phenotype of rev1 and rev3 mutants in yeast has long implicated a functional connection between Rev1 and Pol , although their physical interaction has only been established recently (11)(12)(13)(14). The binding between Rev1 and Pol has been mapped to the C-terminal domain (CTD) of ϳ100 residues of Rev1 and the Rev7 subunit of Pol . Accordingly, the Rev1 CTD has been shown to play an essential role in cell survival following UV irradiation and exposure to DNA-damaging agents such as methyl methanesulfonate or cisplatin in yeast and vertebrate cells (14,15).
In vertebrates, but not in lower eukaryotes such as Saccharomyces cerevisiae, the Rev1 CTD has also been shown to interact with disordered Rev1-interacting regions (RIRs) of Y-family polymerases , , and (12,13,16,17). Such interactions are functionally important for Pol and Pol activity in translesion synthesis. For example, the protective effect of Pol against benzo[a]pyrene in mammalian cells requires the binding of the Pol RIR and Rev1 (18,19); in addition, the Rev1-Pol RIR interaction promotes nuclear accumulation of Rev1 at sites of UV irradiation and helps suppress spontaneous mutations in human cells (20).
Despite its essential role in translesion synthesis, there has been no structural information about the Rev1 CTD or its complex with other translesion polymerases. As a first step toward understanding the molecular basis of the Rev1-mediated assembly of translesion polymerase complexes, we have determined the solution structures of the mouse Rev1 (mRev1) CTD and its complex with the RIR of mouse Pol (mPol ), and have mapped its binding interface toward the Rev7 subunit of Pol using yeast two-hybrid assays. Our structural and biochemical studies reveal two distinct but adjacent binding surfaces of the mRev1 CTD that separately interact with Pol and the RIRs of insertion polymerases in translesion synthesis.

EXPERIMENTAL PROCEDURES
Molecular Cloning and Protein Purification-The NMR constructs contain the mRev1 CTD of 100 residues (1150 -1249) or 115 residues (1135-1249) cloned into a modified pMAL-C2 vector (New England Biolabs) to yield a His 6 -MBP-tagged protein with a TEV site between MBP and the Rev1 CTD. The mPol RIR constructs of 546 -616 and 560 -582 were cloned into a modified pET15b vector (EMD Biosciences) to produce His 10 -GB1-tagged protein with a TEV site between GB1 and the RIR.
The His 6 -MBP-tagged Rev1 CTD was overexpressed in GW6011 Escherichia coli cells, induced with 0.1 mM isopropyl 1-thio-␤-D-galactopyranoside at 18°C for 18 h, and purified by nickel-nitrilotriacetic acid affinity chromatography. After TEV digestion to remove the His 6 -MBP tag, the Rev1 CTD was further purified by size-exclusion chromatography. The mPol RIR was overexpressed in BL21(DE3)STAR E. coli cells, induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside at 37°C for 6 h, and purified following a similar procedure as the mRev1 CTD. The mRev1 CTD-Pol RIR complex was pre-pared by co-purification. Isotopically enriched proteins were overexpressed in M9 media using 15 N-NH 4 Cl and 13 C-glucose as the sole nitrogen and carbon sources (Cambridge Isotope Laboratories). NMR buffers contain 25 mM sodium phosphate, 100 mM KCl, and 10% D 2 O or 100% D 2 O (pH 7.0).
NMR Spectroscopy-NMR experiments were conducted using Agilent INOVA 600 or 800 MHz spectrometers at 25°C and 37°C for the mRev1 CTD and the mRev1 CTD-Pol RIR complex, respectively. Backbone resonances were assigned based on standard three-dimensional triple-resonance experiments (21), and side chain resonances were assigned using sparsely sampled high-resolution four-dimensional HCCH-TOCSY and four-dimensional HCCONH TOCSY experiments (22). Distance constraints were derived from high-resolution three-dimensional 15 N-or 13 C-separated NOESY-HSQC experiments and from sparsely sampled four-dimensional 13 C-HMQC-NOESY-15 N-HSQC and four-dimensional 13 C-HMQC-NOESY-13 C-HSQC experiments. NMR data were processed by NMRpipe (23) and analyzed with Sparky (24). NOE cross-peaks were analyzed with a combination of manual and automated assignments and converted into distance constraints using the calibration module in CYANA (25). Dihedral angles were derived from TALOSϩ analysis of chemical shift information (26) and from analysis of local NOE patterns. Structural ensembles were generated with CYANA (25). The final structural ensembles (20 structures) of the mRev1 CTD and the mRev1 CTD-Pol RIR complex display with no NOE violations Ͼ0.5 Å and no dihedral angle violations Ͼ5°. The quality of these structures can be evaluated in Tables 1 and 2.
Yeast Two-hybrid Analysis-Protein-protein interactions in the yeast two-hybrid system were performed in the PJ69 -4A strain of yeast (27). The mRev1 CTD(1150 -1249) and mRev7 harboring the previously described R124A substitution (28) were cloned into the pGAD-C1 (GAL4 activation domain) and pGBD-C1 (GAL4 DNA-binding domain) plasmids marked with leucine and tryptophan, respectively. The assay was performed by growing strains harboring the two plasmids in 3 ml of media lacking leucine and tryptophan for 2 days at 30°C and spotting 5 l of cells on selective medium plates lacking leucine and tryptophan (ϪLW) and on medium also lacking adenine and histidine (ϪAHLW) to score positive interactions. Interactions were scored after 3 days of growth at 30°C. Site-directed mutations were generated using the QuikChange protocol (Stratagene) and verified by sequencing.

RESULTS
Rev1 CTD Adopts Atypical Four-helix Bundle-After extensive screening for Rev1 CTD constructs, we have identified the mRev1 CTD(1150 -1249) with a cleavable His 6 -MBP tag as the optimal construct for structural characterization by NMR. With the exception of disordered residues at the N terminus, the mRev1 CTD is well structured, with mean pairwise r.m.s.d. of 0.47 and 0.94 Å for the backbone and heavy atoms, respectively (Fig. 1A). The detailed statistics on the structural ensemble are given in Table 1.
The structure of the mRev1 CTD contains an atypical fourhelix bundle consisting of mixed parallel and anti-parallel helices (Fig. 1B). Starting from ␣1 (Phe 1163 -Thr 1175 ) in a down-ward orientation and connected by a long, seven-residue loop, ␣2 (Glu 1183 -Glu 1197 ) is positioned to the left of ␣1 in an upward, anti-parallel orientation. Followed by a short threeresidue loop across space, ␣2 connects into a downward oriented ␣3 (Leu 1201 -Gln 1217 ) located adjacent to and packed parallel with ␣1. Helices ␣3 and ␣4 (Val 1222 -Thr 1241 ) are connected by a short loop of four residues, with ␣4 positioned adjacent to ␣2 and ␣3 and pointing upward. The longest helix ␣4 is tilted at a small angle of ϳ20°with respect to ␣2 and ␣3 and is oriented largely parallel with ␣2 and anti-parallel with ␣3.
Similar to the formation of other four-helix bundles, packing of the mixed parallel and anti-parallel four-helix bundle of the mRev1 CTD involves an interaction network of hydrophobic residues among individual helices, including Phe 1163 , Val 1166 , Leu 1170 , Trp 1173 , Ile 1174 of ␣1, Ile 1185 , Val 1188 , Val 1189 , Tyr 1191 , Cys 1192 , Leu 1195 , Ile 1196 of ␣2, Leu 1201 , Leu 1204 , Val 1207 , Ile 1208 , Met 1211 , Leu 1214 , and Met 1215 of ␣3, and Trp 1223 , Phe 1227 , Leu 1231 , Val 1234 , and Leu 1238 of ␣4 that collectively form an extended hydrophobic core (Fig. 1B). In addition to the central four-helix bundle, the residues C-terminal to ␣4 form an extended ␤-loop, with the side chains of Leu 1246 and Val 1248 juxataposed to interact with the exposed hydrophobic surface between ␣3 and ␣4. The vast majority of these hydrophobic residues are highly conserved from yeast to human (Fig. 1C), supporting the existence of a stable protein module in the Rev1 CTD across different species.
Intriguingly, compared with typical four-helix bundle proteins containing equally spaced helices, the location of ␣1 in the mRev1 CTD is much closer to ␣3 than to ␣2. This creates an extended, solvent exposed hydrophobic surface that is optimal for interaction with other proteins. Query of the Dali Server (29) did not reveal a similar fold of isolated four-helix bundles with r.m.s.d. Ͻ4 Å or Z-score Ͼ5. However, a similar topology can be found as part of seven-helix bundle proteins (e.g. CID of PCF11, Protein Data Bank code 2BF0) to interact with other structural elements, reinforcing the notion that the Rev1 CTD is ideally suited as a scaffolding protein.
Rev7 Recognition Surface Centers at ␣2-␣3 Loop and ␣3 Helix of Rev1 CTD-Having elucidated the structure of the mRev1 CTD, we next probed its interaction with the Rev7 subunit of Pol using yeast two-hybrid assays. The mRev1 CTD and mouse Rev7 (mRev7) containing a R124A substitution, thought to stabilize its closed conformation (28), were fused to either the activation domain or the DNA-binding domain of the Gal4 transcription factor, respectively. Reciprocally, we also appended the mRev1 CTD to the DNA-binding domain and mRev7 to the activation domain of Gal4. The presence of the constructs after transformation into the PJ69-4A strain of yeast was verified by growth on selective medium plates lacking leucine and tryptophan (ϪLW). Interactions between the two proteins activate the expression of the HIS3 and ADE2 reporter genes, both driven by promoters responsive to the Gal4 transcription factor. These interactions were scored by growth on selective medium plates additionally lacking adenine and histi-   (12), the interaction between the mRev1 CTD and mRev7 resulted in robust growth of strains in selective media lacking AHLW when either protein was fused to the activation domain or DNA-binding domain of Gal4; in contrast, no growth was observed in strains expressing the mRev1 CTD and empty expression plasmids, verifying that the Rev1 CTD-Rev7 interaction is specific ( Fig.  2A).
After establishing a strong interaction between the mRev1 CTD and mRev7, in an effort to identify "hot spot" residues that contribute prominently to the binding energy, we mutated pairs of residues of the mRev1 CTD and evaluated their interactions with mRev7 in yeast two-hybrid assays using the mRev1 CTD fused to the DNA-binding domain and mRev7 appended to the activation domain of Gal4.
We first evaluated the effect of mutating pairs of highly conserved hydrophobic residues of individual helices of the mRev1 CTD (Fig. 2B). Double mutations of V1166A/L1170A within ␣1, I1185A/V1189A of ␣2, V1207A/M1211A of ␣3, or W1223A/F1227A of ␣4 abrogated the mRev1 CTD interaction with mRev7. Because these residues form the hydrophobic core of the mRev1 CTD, their substitutions by alanine likely disrupted the proper folding of the mRev1 CTD and thus its interaction with mRev7.
We next evaluated the contribution of surface exposed hydrophilic residues of individual helices toward Rev7 binding, using double alanine substitutions or mutations that result in single-or double-charge reversion (Fig. 2, B-D).  had little effect on Rev7 binding, substitutions of positively charged Lys 1209 /Arg 1213 of ␣3 with negatively charged glutamate residues resulted in a significant reduction of the mRev1 CTD-Rev7 binding (Fig. 2, C and D). Interestingly, mutation of K1209E, but not R1213E, of ␣3 severely compromised the interaction with Rev7 in yeast two-hybrid assays, suggesting that Lys 1209 in ␣3 of the mRev1 CTD is directly involved in Rev7 binding.
To more specifically delineate mRev1 CTD residues important for Rev7 binding, we engineered a set of mutations that span the ␣2-␣3 loop and ␣3 of the mRev1 CTD and tested their ability to disrupt the Rev7 interaction (Fig. 3A). Mutations of hydrophobic residues (L1201A or L1204A) involved in packing of the four-helix bundle disrupted the mRev1 CTD-Rev7 interaction, whereas mutations of Y1210F and L1206A had no effect. Mutation of R1213D, a surface exposed hydrophilic residue at the C-terminal end of ␣3 also did not affect the mRev1 CTD-Rev7 interaction, suggesting that the mRev1 CTD recognition by Rev7 is mediated by the N-terminal half of ␣3 of the mRev1 CTD. Consistent with this notion, single amino acid substitutions of hydrophilic residues at the N-terminal part of ␣3 containing an opposite charge (E1202K, K1203E, or D1205R) completely abolished the Rev7 interaction. Additionally, point mutations of K1199E and D1200R located in the loop connecting ␣2 and ␣3, either significantly reduced or abolished the Rev7 interaction. No binding was observed in control experiments between the mRev1 CTD harboring these mutations and an empty expression plasmid, verifying the specificity of these interactions in our assays. Taken together, these results define the ␣2-␣3 loop and the N-terminal half of ␣3 of the Rev1 CTD as the primary Rev7-binding site (Fig. 3B).
A Minimal RIR of Pol for Rev1 Interaction-The interactions between vertebrate Rev1 and other members of the Y-family polymerases have been mapped to the Rev1 CTD and disordered RIR fragments of ϳ50 residues within Pol , Pol , and Pol (17). Because the RIR of human Pol has the tightest binding affinity toward the Rev1 CTD (19), its mouse counterpart was chosen for characterization by NMR. The mPol RIR(546 -616) displays a typical 1 H-15 N HSQC spectrum of a disordered peptide, with amide resonances showing a narrow chemical shift distribution. By analyzing mPol RIR amide resonances that experience substantial perturbations upon binding to the mRev1 CTD, we have identified a 23-residue RIR peptide of mPol (560 -582) that not only binds to the mRev1 CTD tightly but also causes identical resonance perturbations for the mRev1 CTD as the longer peptide. This 23-residue peptide was subsequently used to investigate the mRev1 CTD-Pol RIR interaction.
Binding-induced Folding of mRev1 CTD and Pol RIR-During our studies of the mRev1 CTD, we found that extending the ϳ100 residue mRev1 CTD construct by 15 residues at the N terminus improved the sample yield and stability. This optimized construct (mRev1(1135-1249)) was used for structural determination of the mRev1 CTD-Pol RIR complex. The overall structure of the mRev1 CTD-Pol RIR complex is well defined, with mean pairwise r.m.s.d. of 0.42 and 0.89 Å for the backbone and heavy atoms, respectively (Fig. 4A). The detailed statistics on the structural ensemble are given in Table 2.
The core helix-bundle structure of the mRev1 CTD in complex with the Pol RIR is similar to that of the free protein. However, the six residues N-terminal to ␣1 (Asn 1156 -Val 1161 ) are completely disordered in the free Rev1 CTD, whereas binding of the Pol RIR induces folding of these residues into a ␤-hairpin that projects over the shallow hydrophobic surface between ␣1 and ␣2 and creates a deep hydrophobic cavity for high-affinity interaction with the essential FF motif of the Pol RIR (Fig. 4, B-D).
The binding of the mRev1 CTD similarly induces folding of the disordered Pol RIR into a three-turn ␣-helix, starting from Phe 566 and ending at Ile 575 (Fig. 4). The folding of the RIR helix is stabilized by a prototypical N-helix cap at Ser 565 (30), with the side chain of Ser 565 forming hydrogen bonds with amides of Phe 567 and Asp 568 and its backbone carbonyl oxygen atom forming another hydrogen bond with the amide of Lys 569 . Substitution of the corresponding serine residue in human Pol RIR by alanine reduced its binding affinity toward Rev1 by more than 50% in a yeast two-hybrid assay (19); however, substitution of serine with proline, another N-helix cap residue, did not affect Rev1 binding (19), highlighting the important contribution of an N-terminal cap to the stability of this binding-induced RIR helix.
Immediately following Ser 565 are two essential phenylalanine residues Phe 566 and Phe 567 that emanate from the same side of the RIR helix to interact with the Rev1 CTD. The aromatic ring of Phe 566 extends over a hydrophobic surface patch formed by residues Trp 1173 , Thr 1176 , and Ile 1177 located at the C-terminal end of ␣1 and the loop immediately following it. The side chain of Phe 567 , with its phenyl ring located almost perpendicular to that of Phe 566 , wedges into and is completely immersed in a deep hydrophobic pocket of the Rev1 CTD. The base of the hydrophobic pocket is formed by Leu 1170 of ␣1 and Val 1188 of ␣2. At one side of the hydrophobic pocket lies the indole ring of the Trp 1173 of ␣1, whereas the opposite side of the pocket is sealed by the side chains of Leu 1157 and Ala 1158 that project over from the newly forged ␤-hairpin in response to the binding of the Pol RIR. These core hydrophobic interactions are additionally supported by interactions between Phe 567 and the side chain methylene groups of the nearby Gln 1187 of ␣2. Reflecting their essential role in the Rev1 CTD-RIR interaction, the FF motif is strictly conserved, their strong interactions with the Rev1 CTD are supported by the observation of numerous intermolecular NOEs (Fig. 4E), and alanine substitution of either of the FF residues abolishes the Rev1 CTD-RIR interaction (19).
The two residues after Phe 567 , Asp 568 , and Lys 569 are surfaceexposed and are not engaged in Rev1 CTD interaction. In contrast, the following residue, Lys 570 is located on the same side of the RIR helix as the two essential phenylalanine residues, with its side chain extending over the aromatic ring of Phe 566 . A number of intermolecular NOEs can be observed between the side chains of Lys 570 of the Pol RIR and Glu 1172 of the mRev1 CTD; additional NOEs between both residues to the aromatic ring of Phe 566 in the Pol RIR are also visible, suggesting that these two residues are located in the vicinity of Phe 566 and they are engaged in a specific charge-charge interaction. Reflecting this observation, Lys 570 is highly conserved among vertebrate RIRs that have been shown to interact with the Rev1 CTD.
In addition to these essential and highly conserved interactions, intermolecular NOEs have also been observed between Pol RIR residues Lys 564 , Arg 571 , and Arg 574 and mRev1 CTD residues Met 1181 , Ala 1158 , and Ala 1160 , respectively. Although Conserved residues are colored, with the essential FF motif highlighted. ŝ refers to a helix cap residue that is predominantly serine but can be replaced by proline. E, representative intermolecular NOEs (indicated by arrows) between the aromatic side chains of Phe 566 and Phe 567 of the mPol RIR and their interacting residues of the mRev1 CTD. these interactions may provide additional support to anchor the Pol RIR onto the Rev1 CTD, these RIR residues are not conserved, and they are unlikely to contribute significantly to the binding affinity.
Our structure of the mRev1 CTD-Pol RIR complex has revealed a binding surface of the Rev1 CTD that consists of ␣1 and ␣2 helices and a newly forged N-terminal ␤-hairpin loop in response to RIR interaction. This structurally defined RIRbinding surface is located adjacent to, but is distinct from, the Rev7-binding surface of the Rev1 CTD centered at the ␣2-␣3 loop and the N-terminal half of the ␣3-helix as revealed by our yeast two-hybrid studies.

Conserved and Acquired Roles of Rev1 CTD in Translesion
Synthesis-Rev1 is a unique member of the eukaryotic Y-family polymerases and is conserved from yeast to human. Its gene was identified in a screen for reversionless mutants of S. cerevisiae after UV irradiation (6), and it was the first Y-family polymerase that was characterized enzymatically (31). The limited polymerizing ability of Rev1 (31), coupled with the profound effect of the rev1-1 mutation, which did not affect the catalytic activity of Rev1, led to the proposal of a "second function" of Rev1 in translesion synthesis (32). This second function is now attributed to the scaffolding function of the Rev1 CTD that coordinates recruitment of Pol (14,33). Deletion of the Rev1 CTD completely abolishes Rev1-dependent translesion synthesis; likewise, elevated levels of the Rev1 CTD show a strong dominant-negative effect on cell viability and induced mutagenesis after DNA damage in yeast (14). Recent studies have revealed a similarly important function of the Rev1 CTD in higher eukaryotes and suggest that the vertebrate Rev1 CTD may have acquired a more prominent role in recruiting Pol and controlling translesion synthesis in the absence of monoubiquitinated PCNA at the stalled replication fork (15,34). Taken together, these results highlight an essential and highly conserved role of the Rev1 CTD-Pol interaction in translesion synthesis that has now been mapped to the ␣2-␣3 loop and the N-terminal half of the ␣3-helix in the Rev1 CTD.
In contrast to the Rev1-Pol interaction that is conserved in all eukaryotes, the Rev1 interaction with the RIRs of Pol , , and has only been found in vertebrates, but not in S. cerevisiae or Schizosaccharomyces pombe, suggesting an evolutionary divergence between animals and yeast (17). Because translesion synthesis accounts for Ͻ10% of damage bypass events in yeast (35), whereas the frequency of potentially mutagenic translesion synthesis may be as high as 50% in higher eukaryotes (36), it is possible that higher eukaryotic Rev1 have acquired additional interactions with other Y-family polymerases outside the essential Rev7 binding surface to achieve a better regulation of translesion bypass. In this regard, several studies have suggested that in higher eukaryotes, translesion synthesis can be carried out in a PCNA ubiquitination-independent manner (34,37,38). In the absence of monoubiquitinated PCNA, which enhances the residence time of translesion polymerases at the replication fork (39), recruitment of the Y-family polymerases , , and may particularly benefit from their RIR-mediated interactions with Rev1, which binds independently to PCNA or primer-template junctions of stalled replication forks. The acquired interaction of translesion polymerases , , and with the Rev1 CTD may also facilitate the transition from an insertion polymerase complex to the extension polymerase complex of Rev1-Pol after base insertion. Alternatively, given that vertebrates have four Y-family polymerases, each specializing at bypassing a specific type of lesion with relatively high accuracy and efficiency, it is tantalizing to speculate that another function of the Rev1 CTD-RIR interaction is to accelerate the exchange to the correct insertion polymerase if the initially recruited insertion polymerase cannot efficiently bypass the DNA lesion to reduce the error rate of translesion synthesis.
Specificity of Interaction between Rev1 CTD and RIR-Our structure of the mRev1 CTD-Pol RIR complex has revealed a surprisingly small binding interface, with the Pol RIR forming a short ␣-helix spanning only ten residues. Apart from Phe 566 , Phe 567 , and Lys 570 of the RIR helix that form specific interactions with the Rev1 CTD and Ser 565 that serves as an N-terminal helix cap (Fig. 4D), the remaining residues are highly variable, and they can be substituted by alanine without affecting the binding affinity toward the Rev1 CTD (19). Interestingly, Lys 570 can also be substituted by alanine without an obvious effect on binding affinity (19), suggesting that Lys 570 , despite forming a charge-charge interaction with Glu 1172 of the mRev1 CTD, does not contribute significantly to the binding energy.
In contrast, the stability of the RIR helix has a significant impact on its binding affinity toward the Rev1 CTD. Substituting the helix cap Ser 565 by alanine significantly reduced the Rev1 CTD-RIR binding (19); likewise, proline substitution of RIR residues after the FF motif (D568P, K569P, K570P, or R571P) abolished the Rev1 interaction (19), suggesting that the formation of the RIR helix requires a minimum of six residues in addition to a helix stabilizing cap. Such an observation may account for a lack of binding affinity of the Rev1 CTD toward the FF motif found in the PCNA-interacting peptides of Pol and Pol (40). Because the PCNA-interacting peptide of Pol contains a single residue after the FF motif, and the PCNAinteracting peptide of Pol contains a proline residue at the second position after the FF motif, neither is expected to be an effective binding partner of the Rev1 CTD.
Targeting Rev1 CTD-mediated Translesion Synthesis for Cancer Therapy-A hallmark of cancer cells is the accumulation of a vast number of mutations in their genomes (41)(42)(43). Cancer-specific mutations have been shown to increase the capacity of cancer cells to carry out mutagenic translesion bypass (44,45), and extensive mutagenesis in turn contributes to the development of drug resistance in relapsed tumors after chemotherapy (46). The recent discovery that depletion of Rev1 greatly reduces the number of carcinogen-induced lung tumors in mice highlights an important role of translesion synthesis in cancer development (47). In human ovarian carcinoma cells, alteration of the Rev1 level modulates the cytotoxicity and mutagenicity of cisplatin (48,49). Depletion of Rev1 or Rev3L, the catalytic unit of mouse Pol , sensitizes B-cell lymphomas and chemoresistant lung adenocarcinomas to cisplatin treatment in vivo (50). Suppression of Rev1 limits cyclophosphamide-induced mutagenesis in vitro and delays the emergence of chemoresistant tumors in vivo (50,51). These studies high-light the therapeutic potential of inhibiting translesion synthesis in cancer treatment.
Given the essential function of the Rev1 CTD in translesion synthesis, compounds that transiently disrupt the Rev1 CTD-Pol interaction could sensitize cancer cells to DNA damaging therapeutic agents while reducing their mutagenic consequences, suggesting that these compounds may be developed into novel adjuvants to enhance the clinical outcome of current therapies. In this regard, the elucidation of the Rev1 CTD structure and its binding surfaces toward Rev7 and the Pol RIR is an important first step toward this goal. Our structural studies have revealed an atypical four-helix bundle fold of the mRev1 CTD, and our yeast genetic data have further identified a set of hot spot residues of the Rev1 CTD and have provided the first residue-specific view of the Rev7-binding interface of the Rev1 CTD, laying a molecular framework for designing novel inhibitors to disrupt the essential and evolutionary conserved Rev1 CTD-Pol interaction. The discovery of multifaceted recognition of the Rev1 CTD by Rev7 and RIR has hinted at the exciting possibility of generating potent inhibitors that separately occupy the Rev7-or RIR-binding surfaces of the Rev1 CTD, which can be used individually or in combination to suppress translesion synthesis. Should these compounds display the desired effectiveness in vivo, they could become a novel class of therapeutics to sensitize cancer cells to chemotherapy and to suppress the development of cancer drug resistance.