Structural insights into the assembly of human translesion polymerase complexes

Abstract

In addition to DNA repair pathways, cells utilize translesion DNA synthesis (TLS) to bypass DNA lesions during replication. During TLS, Y-family DNA polymerase (Polη, Polκ, Polι and Rev1) inserts specific nucleotide opposite preferred DNA lesions, and then Polζ consisting of two subunits, Rev3 and Rev7, carries out primer extension. Here, we report the complex structures of Rev3-Rev7-Rev1^CTD and Rev3-Rev7-Rev1^CTD-Polκ^RIR. These two structures demonstrate that Rev1^CTD contains separate binding sites for Polκ and Rev7. Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1. We also verified through FRET experiment that Rev1, Rev3, Rev7 and Polκ form a stable quaternary complex in vivo, thereby suggesting an efficient switching mechanism where the “inserter” polymerase can be immediately replaced by an “extender” polymerase within the same quaternary complex.

Graphical abstract

Keywords

translesion DNA synthesis / Rev1 / Polκ / Polζ / complex structure

INTRODUCTION

DNA lesions occurring during replication often stall the replicative DNA polymerases, block DNA synthesis and result in genome instability. One solution adopted by cells is to replace the stalled replicative DNA polymerases by specialized translesion DNA synthesis (TLS) polymerases (Goodman, 2002; Prakash et al., 2005; Lehmann et al., 2007; Guo et al., 2009; Sale et al., 2012), which are able to replicate the DNA across the damaged site. Human TLS polymerases include four Y-family DNA polymerases (Polη, Polκ, Polι and Rev1) that exhibit high substrate flexibility, low fidelity and limited proofreading ability (Goodman, 2002; Prakash et al., 2005; Guo et al., 2009; Sale et al., 2012), and one B-family DNA polymerase (Polζ) that consists of two protein subunits, namely Rev3 and Rev7 (Lawrence and Hinkle, 1996; Zhu and Zhang, 2003; Gan et al., 2008).

The current model of TLS has two steps as follows. In the first step, after the monoubiqutination of proliferating cell nuclear antigen (PCNA), Y-family TLS polymerases are recruited to the DNA lesions to replace the stalled replicative DNA polymerase and insert specific nucleotides opposite certain DNA lesions (Edmunds et al., 2008; Chen et al., 2010; Freudenthal et al., 2010). Polη, Polκ and Polι possess at least one Ub-binding domain (UBM or UBZ), a PCNA-interacting peptide (PIP), and a Rev1-interacting region (RIR) (Bienko et al., 2005; Plosky et al., 2006; Bomar et al., 2010). Rev1 is a multiple domain polymerase that sequentially contains an N-terminal BRCT domain that interacts with PCNA, a catalytic domain, two UBMs and a C-terminal polymerase-interacting domain (Acharya et al., 2006; Guo et al., 2006; Auerbach and Demple, 2010). Because of their Ub-binding and PCNA-interacting domains, Y-family TLS polymerases with a higher affinity for ubiquitinated PCNA assemble at the damaged template. In the second step of TLS, Y-family TLS polymerases are switched to Polζ under the regulation of Rev1, and then Polζ extends a few additional nucleotides before a replicative polymerase restarts normal DNA replication (Guo et al., 2001; Zhu and Zhang, 2003; Lehmann et al., 2007; Gan et al., 2008; Andersen et al., 2011). The C-terminal polymerase-interacting domain of Rev1 (Rev1^CTD) is able to bind to the other three Y-family TLS polymerases (Guo et al., 2003; Ohashi et al., 2004; Friedberg et al., 2005; Kosarek et al., 2008; Ito et al., 2012) as well as Rev7, which is the accessory subunit of Polζ (Murakumo et al., 2001; Masuda et al., 2003; Acharya et al., 2005). Thus, Rev1^CTD plays a crucial role in switching Y-family TLS polymerases to B-family TLS polymerase. However, the detailed molecular mechanism is still unclear.

Polη, ι and κ interact with Rev1^CTD through their RIR motifs, which have been mapped by mutational analysis and multiple RIR truncations to approximately 20 residues within Polη, ι and κ (Guo et al., 2003; Ohashi et al., 2004; Kosarek et al., 2008; Guo et al., 2009; Ohashi et al., 2009). Two consecutive phenylalanines (FF) are essential and conserved in all RIRs (Ohashi et al., 2009). Regarding TLS polymerase selection, it remains unclear whether factors other than the lesion itself, such as the interactions among Rev1 and Polη, ι and κ, offer specificity for the cognate polymerase to be recruited to a specific lesion.

Rev1-Polζ complex is not only indispensible for most translesion DNA synthesis events (Shachar et al., 2009; Livneh et al., 2010) but also functions in many DNA repair pathways (Okada et al., 2005) such as DNA interstrand crosslink repair (Räschle et al., 2008) and homologous recombination repair (Sharma et al., 2011). Studies have increasingly indicated that defects in REV3 and REV1 genes are closely related to the development of tumors and the drug resistance of cancer cells (Lin et al., 2006; Dumstorf et al., 2009; Doles et al., 2010; Xie et al., 2010). Moreover, the Fanconi anemia pathway regulates translesion synthesis activity through an interaction between monoubiquitinated Rev1 and the Fanconi anemia core complex (Kim et al., 2012).

Despite a series of structures of the catalytic domain of Y-family TLS polymerases (Nair et al., 2004; Uljon et al., 2004; Nair et al., 2005; Alt et al., 2007; Biertümpfel et al., 2010) and Rev7-Rev3 structure (Hara et al., 2010) have been reported, little is known about the assembly of Rev1 with Polζ and other Y family polymerases. Here, we present the complex structures of human Rev3-7-1 and Rev3-7-1-Polκ. Our structures show that Rev1 contacts RIR motif of Polκ via a hydrophobic pocket formed by its N-terminal β-hairpin, α1, α2 helices and α1-α2 loop. In addition, we verified the details of Rev7-1 interface indicated by crystal structures using an array of pull-down assays, and confirmed the function of Rev3 in inducing and stabilizing the TLS machinery through BIAcore experiments. Moreover, a structural comparison between previous structures (Pozhidaeva A, 2012; Wojtaszek et al., 2012b) and ours show that Polη and Polκ essentially bind to Rev1^CTD in identical manner so that the Rev1 interaction seems to have little effect on polymerase selection. These observations, together with our FRET results, suggest that Rev1, Rev3, Rev7 and Polκ form a stable quaternary complex in vitro and in vivo, and provide comprehensive insight into TLS polymerase switching mechanism that is mediated by Rev1^CTD interactions.

RESULTS AND DISCUSSION

Crystal structure of the Rev3-7-1 complex

Because the free Rev1^CTD was susceptible to heavy degradation, it was challenging to obtain stable human Rev3-7-1 samples. Therefore, a 5(Gly-Ser)-linker sequence was introduced between full-length Rev7 and Rev1^CTD sequences to obtain Rev7 and Rev1 fusion protein (Rev7-Rev1^CTD) (Janda et al., 2010). Rev7 Arg124 was also mutated to Ala as previously reported (Hara et al., 2010). Then, Rev7-Rev1^CTD protein was co-expressed with the Rev7 binding domain of Rev3 (Rev3^7BD, residues 1847–1898), which allowed us to determine the X-ray crystal structure of the Rev3^7BD-Rev7-Rev1^CTD complex at a resolution of 2.7 Å. For simplicity, we refer to the Rev3^7BD-Rev7-Rev1^CTD complex as the Rev3-7-1 complex.

The overall structure of Rev3-7-1 shows two copies per asymmetric unit (Fig. 1A). The 5(Gly-Ser)-linker, the N-terminal 35 residues segment of Rev1, and the N-terminal 27 residues of Rev3^7BD are disordered and are not built into the final model. In comparison with Rev7-Rev3 complex structure reported previously (Hara et al., 2010), the Rev7-Rev3 structures present a similar conformation except that the loop between β6 and β7′ of Rev7 became visible and ordered in our structure, in which the residues 157–163 formed an α-helix named αD (Fig. 1A). The Rev1^CTD in the Rev3-7-1 complex is a four-helix bundle including parallel and anti-parallel helices α1 (residues 1165–1178), α2 (residues 1184–1200), α3 (residues 1203–1219) and α4 (resides 1223–1244) connected by short loops. Ahead and behind of this four-helix bundle, there are two additional regions including an N-terminal β-hairpin domain (residues 1156–1165) packing against the α1 and α2 helices and a last C-terminal 8-residue tail (Rev1^C-tail, residues 1244–1251) extending across the outward surface of the β8′ and β8′′ sheets of Rev7.

Rev1^CTD binds to the Rev7 molecule at two adjacent interface regions, the C-terminal tail and the α2-α3 loop, resulting in a total buried surface area of approximately 674 Ų (Fig. 2A). The major part interface of Rev7 involves residues from the exposed face of β8′ and β8” sheets, in addition to E101 from the β5 sheet and D138 from the αC-β6 loop, which form numerous hydrophilic and hydrophobic contacts with Rev1′s C-tail and α2-α3 loop. In the Rev1^C-tail-Rev7 interface, five side chain-mediated hydrogen bonds are formed as follows: Y1244 on Rev1^C-tail forms two hydrogen bonds with Q200 on the β8” sheet and E101 on the β5 sheet of Rev7, S1246 on Rev1^C-tail with E204 on the β8” sheet of Rev7, K1249 on Rev1^C-tail with E205 on the β8” sheet and D138 on the αC-β6 loop of Rev7; in addition, two main chain hydrogen bonds are formed between T1247 and K1249 on Rev1^C-tail and L186 on the β8′ sheet of Rev7; another hydrogen bond is formed between the main chain of Y1244 on Rev1^C-tail and E204 on the β8” sheet of Rev7, which make the C-tail of Rev1 tightly interact with Rev7 (Fig. 2B). In the Rev1^{α2-α3 loop}-Rev7 interface, D1202 and E1204 on Rev1^{α2-α3 loop} form salt bridges with T191 and K190 on the β8′ sheet of Rev7, respectively; the main chain of E1200 and K1201 on Rev1^{α2-α3 loop} form hydrogen bond with K198 and Q200 on the β8” sheet of Rev7, respectively; E1204 hydrogen bonds with T191 (Fig. 2C). Moreover, besides these hydrophilic interactions, the residues L1203 on Rev1^{α2-α3 loop}, and Y1244 and L1248 on Rev1^C-tail also form hydrophobic interactions with Rev7 via residues L186, P188 and Y202 (Fig. 2D).

To further test the significance of these Rev1 sites for binding Rev7 and rule out the possibility that the linker produced artificial interacting interfaces between Rev7 and Rev1, several corresponding mutants in Rev7 including L138A, K198A and E205A were performed and their bindings to GST-Rev1^CTD were tested. The corresponding mutants of Rev1^CTD including K1201A, L1203A, Y1244A, K1249A and the C-tail truncation of Rev1^CTD (residues 1130-1243, denoted C8) were also detected in a parallel experiment. Indeed, GST pulldown experiments show that all these mutations remarkably reduced the affinity between Rev7 and Rev1 (Fig. 3C). The pulldown results are in accordance with our structural analyses.

Function of Rev3 in forming and stabilizing the Rev3-7-1 complex

Kodai Hara et al. proposed a Rev7-Rev1 interaction model in which the binding of Rev3 to Rev7 substantially induces a conformation change of Rev7 to create a stable anti-parallel β sheets platform for contacting Rev1^CTD (Mapelli et al., 2007; Hara et al., 2010). Our Rev3-7-1 structure provides additional support for this model by confirming that it is the C-terminal anti-parallel β sheets the interaction region of Rev7 binding to Rev1^CTD. This model was further confirmed through BIAcore experiments. Free Rev1^CTD was immobilized on a CM5 chip. Purified free Rev7 and Rev7-Rev3 complex were examined. Free Rev3 also flowed over immobilized Rev1^CTD as a negative control. BIAcore results proved that the existence of Rev3^7BD significantly increases the affinity between Rev7 and Rev1^CTD (Fig. 3A and 3B), indicating that Rev3 plays a critical role in forming and stabilizing the Rev3-7-1 complex. The requirement of Rev3 to stabilize the Rev1-binding conformation of Rev7 ensures that Rev7 and Rev3 have to be assembled to form a complete Polζ before being recruited by Rev1^CTD to the DNA lesion site.

Crystal structure of Rev3-7-1-Polκ

To create the complex of Rev3-7-1 with Polκ^RIR motif, custom synthesized 10-aa RIR peptide of Polκ was added into hanging drops to soak the crystals overnight prior to data collection. Finally, we obtained the crystal structure of the Rev3-7-1 in complex with Polκ^RIR which we named the Rev3-7-1-Polκ complex, at a resolution of 3.2 Å.

In the Rev3-7-1-Polκ complex, only one of the two Polκ^RIR had ordered electron density and residues spanning 566-572 was constructed into the final model. Residues L1159, A1160 on the N-terminal β-hairpin, L1171, W1175, D1186, V1190 on the α1, α2 helices and I1179, P1182, M1183 on the α1-α2 loop create a large hydrophobic pocket on Rev1^CTD, embracing the two conserved Phe residues (F567, F568) of Polκ RIR peptide which folds into an α-helix upon binding (Fig. 1B). Structure superposition of the Polκ RIR peptide-bound and unbound Rev3-7-1 complex reveals no noticeable structural changes (Fig. 1C), which suggests that binding of RIR motif does not affect the conformation of the Rev3-7-1 complex, and the N-terminal β-hairpin of Rev1^CTD is already folded into a stable structure without binding to the Polκ RIR motif. Furthermore, according to crystal structure analyses, we found that there is no noticeable contact between the N-terminal β-hairpin of Rev1^CTD and the neighboring symmetry molecules (Fig. 4B and 4C), indicating that the formation of the N-terminal β-hairpin of Rev1^CTD is not forced by crystal packing.

Very recently, Wojtaszek et al and Pozhidaeva et al presented NMR structures of mouse Rev1^CTD-Polκ^RIR and human Rev1^CTD-Polη^RIR (Pozhidaeva A, 2012; Wojtaszek et al., 2012b), respectively. Compared with our crystal structures, Polη and Polκ share the same binding region involving the N-terminal β-hairpin, α1, α2 helices and α1-α2 loop of Rev1^CTD, indicating that Polη, ι and κ compete to bind to Rev1 in DNA lesion sites (Fig. 1D). Notably, Rev7 and RIR motifs of Y-family polymerases interact with Rev1 in diagonal positions; meanwhile, the interacting region of Rev1 and Polκ locate opposite to that of Rev1 and Rev7, which leads to minimize steric hindrance considering the large size of these TLS polymerases.

Rev1, 3, 7 and Polκ form a stable quaternary complex in vivo

To confirm the results from the pull-down experiments and crystal structures, formation of Rev3-7-1-Polκ complex in vivo was further characterized using FRET experiment. HEK-293T cells were co-transfected with pcDNA^{Rev3 and Rev7-mKO} and pcDNA^{Rev1 and Polκ-EGFP} as well as pcDNA^{Rev3 and Rev7-mKO} and pcDNA^{PolκRIR-EGFP}, which only differs on the expression of Rev1^CTD. As shown in Fig. 5A, the intensity of Polκ-EGFP fluorescence was determined by both pre- and post- photobleaching of Rev7-mKO. With the expression of Rev1^CTD, photobleaching of Rev7-mKO significantly increased the fluorescence intensity of Polκ-EGFP, thereby indicating robust FRET between these two proteins (Fig. 5A and 5B). By contrast, in the absence of Rev1^CTD, the fluorescence intensity of Polκ-EGFP was not significantly increased after the photobleaching of Rev7-mKO (Fig. 5B), which suggests that Rev7 and Polκ do not directly interact with each other. Therefore, it is the co-expression of Rev1^CTD that leads to the increased FRET signals between Rev7-mKO and Polκ-EFGP , indicating that Rev1^CTD serves as a bridge to assemble Rev7 and Polκ^RIR. Based on our data, Rev3-bound Rev7 and Polκ are able to bind to Rev1 simultaneously to form a stable quaternary complex in vivo, and other types of Y-family TLS polymerases may also assemble into a stable quaternary complex with Rev3-7-1 in a similar manner.

Mechanism of Y-family TLS polymerases switch to Polζ bridged by Rev1^CTD

Upon encountering a damage template, the normal replication machinery stalls at the DNA lesion site, which results in PCNA monoubiquitination that is catalyzed by the Rad6-Rad18 complex (Hoege et al., 2002). Monoubiquitinated PCNA recruits Rev1 and other Y-family TLS polymerases to the damage site (Hoege et al., 2002; Garg et al., 2005; Edmunds et al., 2008; Freudenthal et al., 2010). It has been shown that Rev1 and other Y-family TLS polymerase could been recruited independently (Ito et al., 2012). Meanwhile, Polζ, as a whole, also assembles at the damage site through the interaction between Rev7 and Rev1. As a result, Polζ, Rev1 and Polκ simultaneously assemble to form the stable translesion synthesis machinery, where Rev1 functions as a bridging scaffold. After Y-family polymerase bypasses a number of specific DNA lesions, Polκ is easily replaced by Polζ for the primer extension by the TLS machinery complex mediated by the interaction with Rev1^CTD. In summary, in light of our structural and functional studies, we propose a model of TLS polymerases switching mechanism. This model emphasizes that the “inserter” and “extender” TLS polymerases are loaded together into TLS machinery to facilitate nucleotide insertion and extension as required. When the insertion is completed, the “inserter” polymerase is immediately replaced by the “extender” polymerase in the same complex with Rev1 and may not be required to be stripped from Rev1.

MATERIALS AND METHODS

Protein expression, purification, and crystallization

A sequence encoding a Rev7 and Rev1 fusion protein encompassing full-length human Rev7 with an R124A mutation, 5(Gly-Ser)-linker, and the last 135 amino acids of human Rev1 (Rev1^CTD, residues 1117–1251) was constructed into the MCS1 of pETDuet-1 plasmid with an N-terminal His-tag. A sequence encoding human Rev3 fragment (Rev3^7BD, residues 1847–1898) was cloned into the MCS2 of the same plasmid and coexpressed with Rev7-1^CTD protein in Escherichia coli BL21 (DE3) cells. To purify the Rev3-7-1 complex, the cells were lysed using French press in buffer A containing 50 mmol/L Tris-HCl at pH 8.0, 0.3 mol/L NaCl, 20 mmol/L imidazole, 1 mmol/L PMSF and 0.5% Triton X-100. The ternary complex was purified by Ni-NTA-affinity chromatography (GE Healthcare) and dialyzed to buffer B (20 mmol/L Tris-HCl at pH 8.0, 30 mmol/L NaCl, 5 mmol/L DTT), and then sequentially purified on a Resource Q column and a Superdex 200 gel filtration column (GE Healthcare) in buffer C (20 mmol/L Tris-HCl at pH 8.0, 100 mmol/L NaCl, 5 mmol/L DTT). The eluted fractions were analyzed by SDS-PAGE, and highly purified fractions were pooled and concentrated to ~15 mg/mL for crystallization screening (Hampton Company). The complex crystals were crystallized in 0.1 mol/L sodium citrate at pH 5.8, 1.95 mol/L sodium formate, 20 mmol/L DTT by hanging-drop vapor diffusion at 16°C, and improved by the micro-seeding method.

To obtain the Rev3-7-1-Polκ complex, a custom synthesized 10-aa peptide (KKSFFDKKRS) of the RIR region of Polκ was dissolved in Buffer C at a concentration of 10 mg/mL and added into the hanging drop at a ratio of 1:10 to soak the crystals overnight prior to data collection.

X-ray data collection and structure determination

The diffraction data sets were collected at of Shanghai Synchrotron Radiation Facility (SSRF), on beamline BL17U and processed with HKL2000 software (Otwinowski and Minor, 1997). The crystal belonged to the P2₁2₁2 space group with two copies of the Rev3-7-1 complex per asymmetric unit. The structure was solved by molecular replacement using Molrep of the CCP4 program suite (1994), with the Rev7-Rev3 heterodimer structure (Protein Data Bank [PDB] ID: 3ABD) as the search model (Hara et al., 2010). The resultant high-quality electron density map allows unambiguous building of the Rev1 model. The Rev3-7-1-Polκ structure was solved with the resulting Rev3-7-1 structure as the search model. Model building was performed with Coot (Emsley and Cowtan, 2004) and refinement was carried out using Refmac (Murshudov et al., 1997) and PHENIX (Adams et al., 2010). Data collection and refinement statistics are shown in Table 1. Structure figures were prepared using PyMol (http://www.pymol.org), and the coordinates were deposited in PDB under accession code 4GK0 and 4GK5.

In vitro pull-down assays

His-tagged wild-type and mutant Rev7-Rev3^7BD complex were co-expressed as previously described (Hara et al., 2010). Wild-type and mutant Rev1^CTD (residues 1130–1251) were subcloned into pGEX6p-1 plasmid to produce GST-Rev1^CTD proteins. All of the proteins were overexpressed in Escherichia coli BL21 (DE3). The in vitro binding assays were performed according to standard procedure. Briefly, wild-type or mutant GST-Rev1^CTD -bound glutathione-sepharose beads were incubated with mutant or wild-type Rev7-Rev3 complexes in buffer D (20 mmol/L HEPES at pH 7.5, 150 mmol/L NaCl, 1 mmol/L DTT) for 2 h at 4°C. Protein-bound glutathione resins were washed five times using binding buffer D, and the bound samples were analyzed using Coomassieblue-stained SDS-PAGE. Pull-down assays were repeated three times and data were calculated using ImageJ (National Institute of Health).

BIAcore experiments

Interactions of Rev1^CTD with Rev7 and Rev7-Rev3^7BD were measured on a BIAcore 3000 instrument (BIAcore AB, Uppsala, Sweden). Free Rev1^CTD (residues 1130–1251) was prepared by removing the GST-tag of GST-Rev1^CTD (residues 1130–1251) using prescission protease cleavage, followed by protein purification on a Superdex200 sizing column (GE Healthcare). Rev1^CTD was immobilized on the carboxymethylated dextran surface-modified chip (CM5 chip) in accordance with the amine-coupling protocol of the BIAcore manual. The running buffer (20 mmol/L HEPES at pH 7.5, 150 mmol/L NaCl, and 0.005% [v/v] Tween-20) was filtered through Millipore Film (pore size 0.22 mm) and degassed before use. The binding affinities were evaluated over a range of Rev7-3 (50–1600 nmol/L) and free Rev7 (0.4–12.8 μmol/L) concentrations at 25°C. Meanwhile, for both binding assays, the concentration of 400nmol/L was repeated as an internal control, and the free Rev3 was taken as a negative control. All of the data collected were analyzed using BIAevaluation software version 4.1.

Acceptor photobleaching FRET

FRET experiments were conducted as described by Chen et al (2009). Sequences encompassing Rev3^7BD and Rev7-mKO were simultaneously cloned into a pcDNA vector (pcDNA^{Rev3 and Rev7-mKO}). Similarly, sequences encompassing Polκ^RIR-EGFP (residues 560–615) and Rev1^CTD (residues 1130–1251) were simultaneously cloned into a pcDNA vector (pcDNA^{Rev1 and Polκ-EGFP}). HEK-293T cells were co-transfected with pcDNA^{Rev3 with Rev7-mKO} and pcDNA^{Rev1 and Polκ-EGFP} as well as pcDNA^{Rev3 and Rev7-mKO} and pcDNA^{PolκRIR-EGFP}. We also transfected HEK-293T cells with EGFP fused with mKO (mKO-EGFP) as a positive control, and mKO-Rev7 and EGFP empty vector as a negative control. The FRET experiments were performed using a confocal microscope (FV1000; Olympus) equipped with 488 nm and 515 nm lasers. We used the sequence mode to prevent fluorescence leakage when capturing pre-photobleaching images. The acceptor (mKO) was then bleached by repetitive scanning of the cell with a full-power 515 nm laser for 4 min. Finally, with the same acquisition parameters as those applied to the pre-acceptor photobleaching images acquisition, the post-photobleaching images were acquired. The data were further analyzed using ImageJ software.

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Higher Education Press and Springer-Verlag Berlin Heidelberg 2012