Snapshots of a Y-Family DNA Polymerase in Replication: Substrate-induced Conformational Transitions and Implications for Fidelity of Dpo4

J. Mol. Biol. (2008) 379, 317–330

doi:10.1016/j.jmb.2008.03.038

Available online at www.sciencedirect.com

Snapshots of a Y-Family DNA Polymerase in Replication: Substrate-induced Conformational Transitions and Implications for Fidelity of Dpo4 Jimson H. Wong 1 , Kevin A. Fiala 2 , Zucai Suo 2 and Hong Ling 1 ⁎ 1

Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1 2

Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA Received 3 December 2007; received in revised form 19 February 2008; accepted 19 March 2008 Available online 28 March 2008

Y-family DNA polymerases catalyze translesion DNA synthesis over damaged DNA. Each Y-family polymerase has a polymerase core consisting of a palm, finger and thumb domain in addition to a fourth domain known as a little finger domain. It is unclear how each domain moves during nucleotide incorporation and what type of conformational changes corresponds to the rate-limiting step previously reported in kinetic studies. Here, we present three crystal structures of the prototype Y-family polymerase: apo-Dpo4 at 1.9 Å resolution, Dpo4-DNA binary complex and Dpo4-DNA-dTMP ternary complex at 2.2 Å resolution. Dpo4 undergoes dramatic conformational changes from the apo to the binary structures with a 131° rotation of the little finger domain relative to the polymerase core upon DNA binding. This DNA-induced conformational change is verified in solution by our tryptophan fluorescence studies. In contrast, the polymerase core retains the same conformation in all three conformationally distinct states. Particularly, the finger domain which is responsible for checking base pairing between the template base and an incoming nucleotide retains a rigid conformation. The inflexibility of the polymerase core likely contributes to the low fidelity of Dpo4, in addition to its loose and solvent-accessible active site. Interestingly, while the binary and ternary complexes of Dpo4 retain an identical global conformation, the aromatic side chains of two conserved tyrosines at the nucleotide-binding site change orientations between the binary and ternary structures. Such local conformational changes may correspond to the rate-limiting step in the mechanism of nucleotide incorporation. Together, the global and local conformational transitions observed in our study provide a structural basis for the distinct kinetic steps of a catalytic cycle of DNA polymerization performed by a Y-family polymerase. © 2008 Elsevier Ltd. All rights reserved.

Edited by J. Doudna

Keywords: DNA replication; DNA polymerase; fidelity; conformational transition; little finger domain

Introduction A common challenge to the viability of all living cells is the constant attack by endogenous and exogenous agents that damage genomic DNA. Although various DNA repair pathways exist to

*Corresponding author. E-mail address: [email protected]. Abbreviations used: TLS, translesion DNA synthesis; LF, little finger; Dpo4, DNA polymerase IV; yPolη, yeast DNA polymerase η; dsDNA, double-stranded DNA.

repair DNA lesions, a portion of DNA damage escapes repair and stalls the DNA replication machinery. Translesion DNA synthesis (TLS) helps cells survive DNA damage by replicating through unrepaired lesions. TLS is catalyzed mainly by Yfamily DNA polymerases that are conserved from bacteria to humans.1,2 The translesion DNA polymerases catalyze DNA synthesis past DNA lesions and play a central role in TLS. These TLS polymerases are intrinsically error-prone (low-fidelity) compared to the high-fidelity replicative DNA polymerases in the A- and B-families.3,4 There is no detectable sequence identity between the Y-

0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

318

Snapshots of a Y-Family DNA Polymerase in Replication

family DNA polymerases and the high-fidelity DNA polymerases in the A- and B-families. However, the Y-family enzymes retain the conserved right-handed polymerase core composed of a thumb, palm, and finger domains.5–8 Every Y-family enzyme possesses a unique C-terminal domain in addition to the polymerase core, referred to as the little finger (LF) domain, or alternatively known as the wrist or PAD, in addition to the conserved polymerase core. The LF domain has been shown to increase the overall binding affinity to DNA by contacting the DNA major groove.5,7,9 DNA polymerase IV (Dpo4) from Sulfolobus solfataricus is the most thoroughly studied Y-family polymerase from a structural perspective, and serves as a model system for mechanistic studies of Y-family polymerases. Structural studies of Dpo4 have revealed that the finger and thumb domains of the Y-family polymerases are significantly smaller than the corresponding domains of the A- and Bfamily DNA polymerases. 5 The interactions between these polymerase domains and the DNA substrate at the active site are thus reduced significantly, resulting in a more solvent-accessible and less geometrically restrictive active site in these Y-family polymerases. These structural features enable the low-fidelity Y-family polymerases to accommodate and bypass various DNA lesions and mismatched base pairs in their active sites.10–13 DNA polymerases progress through distinct steps in the catalytic cycle of nucleotide incorporation during DNA replication (Fig. 1).14–17 High-fidelity DNA polymerases bind a primer/template DNA prior to an incoming nucleotide (dNTP) with the finger domain in an open conformation.18 Upon the binding of the dNTP, a high-fidelity DNA polymerase transforms from a binary polymerase-DNA complex into a ternary complex. This process initiates a large conformational change in high-fidelity polymerases whereby the finger domain rotates by 41–46° towards the active site with the O-helix contacting the template base and the incoming dNTP.19,20 This “open-to-closed” conformational change is instrumental in geometrically selecting the correct incoming nucleotide, and has been proposed to be the rate-limiting step in the induced-fit kinetic mechanism.19,20 However, a recent fluorescence study on Klentaq1 shows that the finger domain motion is fast and not rate-limiting.21 DNA polymerases perform nucleotide incorporation using the two metal ion catalysis model to join the 3′OH of the primer strand to the α-phosphate of an incoming nucleotide.22,23 In particular, the activity of Dpo4 was found to be greatly dependent upon

the subtle positioning of its metal ions in the A- and B- metal-binding sites, as well as the specific elemental characteristics of the metal ions.13 In the structures of Dpo4 ternary complexes with undamaged DNA5,13 or DNA containing lesions,10–12 the divalent metal ions were consistently located at the B-site, coordinated by the catalytic side chains of Asp7 and Asp105, and the phosphates of the incoming nucleotide. However, the position of the A-site metal ion was not consistent in these structures. Proper alignment of the incoming nucleotide and the catalytic metal ions within the active site is required for nucleotide incorporation.16,17 Pre-steady state kinetic studies of the catalytic mechanism have been reported for Y-family proteins including archaeal Dpo4,16,24 and yeast DNA polymerase η (yPolη).25 A rate-limiting step for correct nucleotide incorporation has been defined as the step after nucleotide binding and before the chemistry step of nucleotide incorporation (step 3 in Fig. 1). This rate-limiting step was proposed on the basis of kinetic evidence of a slow conformational change step.25,26 However, no previously reported structures of Dpo4 or any other Y-family polymerases reveal a significant conformational change between the binary and ternary complexes in these polymerases.5,27,28 Moreover, no apo-Dpo4 structure is available, which reflects the incomplete nature of the structural analysis regarding the process of DNA polymerization catalyzed by Dpo4 and Y-family enzymes. None of the Y-family polymerases had been characterized structurally in each of the apo-, binary and ternary states. For example, structures of human polymerase κ only include the apo and ternary forms,29,30 while the structures for human pol ι were solved in the binary and ternary forms.28 This raises important questions of whether conformational changes occur during nucleotide incorporation catalyzed by Y-family DNA polymerases and, if so, at what stage(s) of the catalytic cycle do Yfamily DNA polymerases undergo such changes. To address the above questions, we determined the apo-Dpo4 structure at 1.9 Å resolution along with the binary/ternary structures of Dpo4 in complex with a primer/template DNA and a nucleotide at 2.2 Å resolution. These structures exhibit vastly different structural conformations. We also used tryptophan fluorescence spectroscopic analysis to verify the conformational transitions observed in these crystal structures. The conformations of Dpo4 reflect distinct reaction intermediates during nucleotide incorporation, and provide insight into the structural basis of nucleotide incorporation catalyzed by Dpo4 and the Y-family DNA polymerases.

Fig. 1. General kinetic model of nucleotide incorporation by DNA polymerases. Eapo and PPi represent the apo structure of Dpo4 and pyrophosphate, respectively. In step 3, the protein (E) undergoes a rate-limiting conformation change step (E′).

Snapshots of a Y-Family DNA Polymerase in Replication

Results Apo-Dpo4 structure We successfully crystallized the apo-Dpo4 molecule by removing the ten C-terminal residues I342– E351. These ten residues are disordered in the published Dpo4 ternary structures,5,10–13 and in our current DNA complex structures reported here. The truncated Dpo4 possesses full enzymatic activity when compared to the full-length Dpo4 in primer extension assays (data not shown). The apo-Dpo4 crystals diffract to 1.9 Å and have a monoclinic C2 space group, containing one Dpo4 protein molecule in the asymmetric unit (Table 1). The structure was solved by molecular replacement using the type I Dpo4 structure as the search model, which contains a Dpo4, a template/primer DNA and an incoming nucleotide.5 The apo structure exhibits a dramatic difference in conformation when compared to the previously reported ternary structure5 and the binary complex27 in terms of the relative positions of the structural domains (Fig. 2). The published ternary Table 1. Data collection and refinement statistics Crystal A. Data collection Space group Unique reflections Unit cell (a, b, c) a (Å) b (Å) c (Å) β (°) Molecules/AUa Resolution range (Å) Completenessb Rmergec I/Iσ Redundancy B. Refinement statistics Rfreed R-factor Nonhydrogen atomse rmsd from ideal Bond lengths (Å) Bond angles (°) Ramachandran plotf Favored regions (%) Allowed regions (%) Average B-factors (Å2) Protein DNA Metal ions Solvent a

Apo-Dpo4

Complexed Dpo4

C2 28,887

P212121 51,948

92.9 51.1 86.0 98.1 1 20.4–1.92 (1.95–1.92) 93.8 (90.6) 0.043 (0.507) 24.3 (2.0) 3.0 (2.5)

98.0 102.5 106.1 2 24.1–2.20 (2.25–2.20) 97.5 (99.7) 0.055 (0.360) 18.1 (2.1) 2.8 (2.7)

0.247 (4.2) 0.207 2949 (239)

0.256 (4.8) 0.210 7027 (306)

0.014 1.42

0.013 1.56

98.5 100.0

98.0 100.0

46.1

43.2 50.5 41.2 47.5

53.7

AU, asymmetric unit. Data in in parentheses are for the highest resolution set. c Rmerge = ∑|I–〈I〉|/∑I, where I is the integrated intensity of each reflection. d The percentage of data excluded for Rfree definition are denoted in parentheses. e The number of water molecules are denoted in parentheses. f Calculated by Molprobity.55 b

319 and the binary Dpo4 structures have essentially identical overall structures in contrast to the changes observed in the apo form.5,27,31 In the DNA-bound forms, the polymerase core is in a classic righthanded configuration bound to the double-stranded DNA (dsDNA); and the LF domain binds the major groove of the DNA helix (Fig. 2b and c). The major difference between the apo structure and the DNAbound structures is the location of the LF domain (purple) relative to the polymerase core consisting of palm, thumb and finger domains. In the apo structure, the LF domain, which interacts with the finger domain in the DNA-bound forms, is positioned away from the finger domain and associates with the thumb domain (Fig. 2). The movement of the LF domain from the apo-state to the DNAbound states consists of a combination of a 131° rotation and a 1.7 Å translation along the rotation axis (Fig. 2d). This transition results in the LF domain having more contacts with the polymerase core in the apo-form than in the DNA-bound forms. The buried surface (282 Å2) between the LF and thumb domains in the apo form is much larger than the interface (171 Å2) between the LF and finger domains in the DNA-bound Dpo4 structures.5 The large interface in apo-Dpo4 may anchor the flexible LF better in the absence of DNA. Interestingly, the LF domain occupies the DNAbinding cleft, effectively blocking the binding pocket where the duplex DNA would be positioned in Dpo4-DNA complexes. The binding of DNA induces a conformational change that makes the binding cleft available. This movement of the LF domain reveals the structural flexibility of the linker region (residues 234–244 in Dpo4) that tethers the LF finger domain to the polymerase core. In addition to this dramatic rotation of the LF domain, both the thumb and finger domain rotate about 10° into the DNA-binding cleft upon DNA binding, resulting in further “closing” of Dpo4 upon the DNA substrate (Fig. 2d). Each individual domain retains the same structure as the respective domain in the previously reported DNA-bound structures,5,10–13 with rootmean-square deviations (rmsd) of the superposed Cα atoms ranging from 0.3 Å to 0.7 Å. The low rmsd values between corresponding individual domains indicate that the Dpo4 domains rotate as rigid bodies without any rearrangement of secondary structural elements. Although the domain structures are unchanged, the side chains of the residues specifically involved in substrate binding adopt significantly different conformations as Dpo4 transforms from the apostructure to the Dpo4-DNA-dNTP ternary structure (Fig. 3). 5,10–12 In the Dpo4-DNA-dNTP ternary complexes, the nucleotide-binding residues Tyr48 and Arg51 are involved in stabilization of the negatively charged incoming dNTP.5,10,13 Noticeably, the hydroxyl group of Tyr48 is oriented toward the active site and forms a hydrogen bond with the γ-phosphate of the incoming dTTP (Fig. 3d). In contrast, the conserved residues Tyr48 and Arg51 are positioned away from the active site in the

320

Snapshots of a Y-Family DNA Polymerase in Replication

Fig. 2. Crystal structures of Dpo4 in apo, binary and ternary complex forms. All the polymerase structures are depicted to the same scale and the same orientation of their palm domains by superposition of all the structures on the conserved palm domains. The polymerases in (a–c) are in ribbon diagrams and colored with the same color scheme: finger domain, blue; palm domain, red; thumb domain, green; little finger domain, purple; DNA, gray; and dTMP is in sticks. The yellow arrows indicate the region of Dpo4 that contacts the DNA replicating base pair in the active site. (a) Apo-Dpo4, the light blue broken line shows the non-structured loop of the finger domain in the apo-Dpo4. (b) Dpo4-DNA binary complex; (c) Dpo4-DNA-dTMP ternary complex. (d) Superposition of Dpo4 in different forms, apo-Dpo4 (blue), Dpo4DNA binary complex (green), ternary (red). The structures are in Cα traces.

absence of the negatively charged DNA substrate in the apo structure (Fig. 3a). Rotations of the side chains make the nucleotide-binding site more loose and less positively charged in the apo-Dpo4 structure than in a ternary complex (Fig. 4).10 In addition, the apo-Dpo4 structure contains no metal ion in the active site. Interestingly, loop 23 (residues 34–39, broken line in Fig. 2a) in the finger domain of the apo structure is disordered due to the absence of

contacts made by the LF domain and template DNA as observed in the DNA-bound structure.5 Dpo4 binary complex The full-length Dpo4 protein was co-crystallized with a primer/template 14/18-mer DNA substrate in the absence of dNTP. The crystal is in the P212121 space group with two complexes in the asymmetric

Snapshots of a Y-Family DNA Polymerase in Replication

321

Fig. 3. Different side-chain conformations in the nucleotide-binding pocket. The residues (carbon, yellow; nitrogen, blue; and oxygen, red) involved in conformational changes and the nucleotide (carbon, gray) are shown in ball-and-sticks and the rest of the protein is shown in ribbons (gray). The metal ions at the B-site are shown as green spheres. No nucleotide or metal ions were located in the apo-structure. Step-wise conformational changes from the apo-Dpo4 (a), to the binary complex (b), and the dTMP ternary complex (c) in which the dTMP is in trans to the template strand; as well as a standard ternary complex of Dpo4 (Ab-2A, 1S0O10) (d), which contains a dTTP and two catalytic metal ions that are superposed well with the replicative phage T7 DNA polymerase (1T7P) at the active site.

unit. Both Dpo4 molecules are structured only at residues 1–341 and are disordered at the C terminus (residues 342–352) as observed previously.5 The first complex is the Dpo4-DNA binary complex containing Dpo4, a primer/template 14/18-mer DNA duplex and a calcium ion at the active site. The second structure is a ternary complex containing dTMP in the Dpo4 active site, which will be discussed in more detail in the next section. In the binary complex, the domains of Dpo4 retain the same overall structural arrangement as that observed in the Dpo4 ternary complexes reported previously (Figs. 2b and 6a).5,10,11 The aromatic

side chains of Tyr10 and Tyr48 in the Dpo4-DNA binary structure (Fig. 3b) remain in the conformations observed in the apo structure (Fig. 3a), but are in significantly different orientations when compared to the ternary structures (Fig. 3c and d; Table 2). This indicates that a local conformational change has taken place with Tyr10 and Tyr48 upon dNTP binding. Interestingly, Arg51 moves into the active site in the transition from the apo protein to the binary structure in order to provide electrostatic stabilization for the binding of the negatively charged DNA substrate. Dpo4 contacts DNA in the binary complex with a 1530 Å2

322

Snapshots of a Y-Family DNA Polymerase in Replication

interface that stabilizes the overall Dpo4 conformation in which only a weak interaction (only 176 Å2 contact area) exists between the LF and the finger domains.

Table 2. Side-chain torsion angles of Y48 and Y10 in Dpo4 Y48 Structure

χ1

Apo Binary dTMP-ternary Ternary (1S0O)10 Ternary (1JX4)5

174.5 −178.5 −75.4 −73.9 −75.7

Y10 χ2

χ1

χ2

69.9 108.1 87.1 77.8 73.9

−52.5 −34.5 60.1 61.0 55.5

101.0 − 81.2 103.6 109.9 105.9

χ1 = N–Cα–Cβ–Cγ, χ2 = Cα–Cβ–Cγ–Cδ1.

Dpo4-DNA-dTMP ternary complex

Fig. 4. Electrostatic environment and closeness of the nucleotide binding site. Protein is shown as a charged surface where blue and red represent positive and negative potentials, respectively. The DNA and nucleotide are shown as stick models (carbon, gray). The metal ions are shown as green spheres. The arrows point to the site that is different in the three Dpo4 structures: (a) apo-Dpo4 with an incoming nucleotide and metal ions (gray) modeled from Ab-2A into the apo-Dpo4 structure by superimposing the apo-Dpo4 and Ab-2A structures. (b) binary Dpo4DNA complex; (c) standard active site with two metal ions (Ab-2A, 1S0O).10

Unexpectedly, we observed a second molecule in the same asymmetric unit of the complex crystal containing a DNA-bound Dpo4 with a dTMP and a calcium ion in the active site, assembling a mismatched ternary complex. We refer to this complex as the dTMP-ternary complex to distinguish it from previous reported ternary complexes. Interestingly, we intentionally did not include nucleotides in the crystallization conditions in an attempt to crystallize the Dpo4-DNA binary complex. The dTMP arose from the excision of the primer 3′-terminal dTMP of 14/18-mer DNA in co-crystallization. We have confirmed that Dpo4 does possess a weak 3′–5′ exonuclease activity (data shown later). In the dTMP-ternary complex, Dpo4 is structurally identical to the previously published Dpo4 ternary complexes (Figs. 2c and 5a). In the nucleotide-binding site of the complex, Dpo4 maintains side chain conformations of Tyr 48, Arg 51 and Tyr 10 identical with those reported for the Dpo4 ternary structure (Fig. 3c and d; Table 2).5,10 In comparison to the apo form (Fig. 3a) and binary complex (Fig. 3b), Tyr10 rotates out of the active site in the transition to the ternary complex (Fig. 3c and d) in order to accommodate the incoming nucleotide. These conformational changes alter the electrostatic environment in the vicinity of the triphosphate moiety (Fig. 4). In the ternary complexes (Fig. 4c), the nucleotide-binding site is more enclosed and more positively charged than those of the apo and binary structures (Fig. 4a and b). The dTMP pairs with the 15th template base, forming a T:dTMP mismatched base pair in the structure of the dTMP-ternary complex (Fig. 5b). The two T bases are co-planar and well stacked with the neighboring base pair in the DNA duplex as in other regular ternary complexes (Fig. 6a, green). The entire dTMP nucleotide rotates 180° relative to the regular position of an incoming nucleotide observed in a ternary complex and forms a trans base pair with the template T base (Fig. 5). The C1′–C1′ distance (11.4 Å) is comparable with those in the regular geometry. In contrast to a cis base pair in a regular DNA helix, the trans paired dTMP has its αphosphate shifted away from the 3′-end of the primer strand and its sugar O3′ pointed toward to the primer terminus, as observed in a non-productive Dpo4 ternary complex (Fig. 3c).32 The T:T mismatch base pair forms two H-bonds: N–H⋯O

Snapshots of a Y-Family DNA Polymerase in Replication

323

Fig. 5. Watson–Crick and mismatched base pairs. (a) A standard Watson–Crick A:dTTP base pair (cis form) from a Dpo4 ternary complex (Ab-2A, 1S0O).10 (b) T:dTMP mismatched base pair in the dTMPternary complex. The T:dTMP base pair is in a trans form covered with a simulated annealing Fo−Fc omit map contoured at 2.5 σ at 2.2 Å resolution. A water molecule H-bonded with the N3 of dTMP is shown as a red sphere. The H-bonds are shown in blue broken lines with bond lengths labeled.

between N3 and O4 (dTMP) and CH⋯O between O4 and C5A (dTMP) (Fig. 5b). The carbon–oxygen (CH⋯O) hydrogen bond has been observed in both DNA and protein structures, which is weaker than regular H-bonds between N and O atoms but does support base pairing between bases.33,34 Flipping of the dTMP also results in its α-phosphate occupying the position where the β-phosphate of an incoming triphosphate usually resides in the ternary structures (Figs. 3c and d, and 5).11–13 At this position, the α-phosphate is able to make contacts with nucleotide-binding residues Arg51, Thr45, and Asp105, as well as being able to participate in the coordination sphere of the Ca2+ metal ion found at the B position. The phosphate moiety is also in the vicinity of

conserved residues Tyr48 and Lys159 that stabilize the negatively charged moiety in the ternary complexes.13,35 No metal ion is observed in the complex at the A-site, indicating that a proper incoming nucleotide with a triphosphate is required for the metal ion binding at the A-site. The structures of Dpo4 superimpose well between the binary, ternary and the previously reported ternary complex (Fig. 6).5 In particular, the loop (residues 55–59) and the β strand (residues 41–46) of the finger domain) contacting the replicating base pair at the active site do not reposition in the transition between the binary and ternary structures (Figs. 2d and 6a, indicated by arrows). The side chains in the region also keep the identical rotamers.

Fig. 6. Superposition of DNA bound Dpo4 structures. (a) Complex structures in stereo-views, the proteins are in Cα traces and the DNA is in sticks; Dpo4-DNA binary complex (red), dTMP-ternary (green) and type I (gray, 1JX4);5 the red arrows point to the part of the finger domain that shows no movement between the Dpo4 structures. (b) Enlarged DNA structures of the binary (carbon, light blue) and type I ternary (carbon, gray) complexes. The top panel depicts the first three base pairs in the active site oriented identically. The bottom panel represents the first base pair that contacts the finger domain in the active site.

324

Snapshots of a Y-Family DNA Polymerase in Replication

Accordingly, the DNA and dNTP are superimposed well over the binary and ternary structures in the active site (Fig. 6). The DNA and incoming nucleotide align well in terms of the stacking positions and the orientations of the base pairs in the active site (Fig. 6b). In particular, the 3′ ends of the primer in the binary and ternary structures point in an identical direction and locate in the same position at the active site (Fig. 6b). Overall, comparisons of the apo-Dpo4, the binary structure and the ternary complex reveal conformational changes of Dpo4 during nucleotide incorporation, which include a global conformational change upon DNA binding and local conformational changes within the nucleotide-binding site. In contrast, the DNA retains identical conformations in both the binary and ternary complexes in this study. To test the 3′ → 5′ exonuclease activity of Dpo4, we examined the degradation of primer/template by Dpo4 under single turnover reaction conditions at 37 °C. The primer/template (21/41-mer) DNA substrates, containing either a mismatched terminus (A:A) or a matched terminus (T:A), were incubated with Dpo4 to determine if Dpo4 possesses 3′ → 5′ exonuclease activity (see Experimental Procedures). Our results indicate a weak 3′ → 5′ exonuclease actimatch of 5.4(± 0.1) × 10− 7 s− 1 vity for Dpo4 with a kexo −9 −1 mismatch and a kexo of 4(± 2) × 10 s . Although the cleavage occurred slowly, the week-long time requirement for crystal formation should allow Dpo4 to generate enough dTMP that could then, in principle, be used to form the dTMP-ternary complex in the crystals. It is not clear why the excision

rate for the DNA substrate with a matched terminus is higher than that of the mismatched one. However, our previous structural report indicates an analogous phenomenon, in which the weak phosphatase activity of Dpo4 was observed only in the ternary complex with a matched incoming nucleotide and not in the ternary complex with a mismatched incoming nucleotide.5 A double mutant (D105A/ E106A at the active site) has been used as a negative control in these degradation experiments and indicates that mutation of these residues abolishes polymerase activity,5 and eliminates the 3′ → 5′ exonuclease activity of Dpo4. Conformational changes of Dpo4 in solution Tryptophan fluorescence has been used previously to monitor conformational changes in proteins.36,37 Since the wild type Dpo4 protein does not have any intrinsic tryptophan residues, we mutated Tyr274 to tryptophan (Y274W) to generate a fluorescence probe. Tyr274 is located on the LF domain distal to the DNA-binding cleft and is fully exposed to solvent in the DNA-bound Dpo4 structures, but this aromatic residue is buried in the interface between the LF domain and the thumb domain in the apo structure presented here (Fig. 7). The relative location of residue 274 with respect to the DNAbinding site allows us to identify its local environmental changes without interference from direct contacts of the probe with DNA. To probe for conformational changes in the structure of Dpo4 upon DNA binding in solution, we examined the

Fig. 7. Positions of Y274 in the apo and binary complex structures. The protein is in ribbons with the core in cyan and the LF in purple. The DNA is in wheat yellow. The Y274 is in stick bonds and highlighted with yellow carbon atoms. The residues involved in contact with Y274 from the thumb domain are colored orange. (a) apo-Dpo4; (b) binary Dpo4.

Snapshots of a Y-Family DNA Polymerase in Replication

325

Fig. 8. Fluorescence spectra of Dpo4-Y274W. Fluorescence spectra of 2 μM Dpo4-Y274W alone (broken line) and 2 μM Dpo4-Y274W with primer/template DNA (continuous line) added in a 1:1.5 protein to DNA ratio at room temperature.

changes in the tryptophan fluorescence of the Dpo4 Y274W mutant. The fluorescence spectra of the Y274W mutant were collected in the absence and in the presence of primer/template DNA duplexes (Fig. 8). The addition of a 1.5-fold molar excess of DNA (see Experimental Procedures) resulted in a 20% decrease in the fluorescence intensity and a red shift in the peak position from 343 nm to 354 nm. No fluorescence emission was detected in a control experiment consisting of a solution of DNA under the same experimental conditions; so the red shift and fluorescence quenching indicate environmental changes of Trp274 on the LF domain upon DNA binding, where Trp274 shifts from a buried interface to a solvent-exposed protein surface. These results are consistent with the structural observations, in which Tyr274 is shielded against the thumb domain in the apo state and fully exposed when bound by DNA, indicating that the induced conformational change upon binding DNA observed in our crystal structures exists also in solution (Fig. 7).

Discussion Conformational changes in Dpo4, rigidity and flexibility Our crystal structure analysis demonstrates that Dpo4 undergoes both global and local conformational changes as DNA and the incoming nucleotide bind sequentially. The change in the relative orientation and location of the LF domain and the stepwise re-organization of amino acid side chain orientations in the dNTP-binding site constitute the corresponding movement of the protein in different reaction stages of dNTP incorporation. The fluorescence experiment used to identify changes in the tryptophan environment confirms that the conformational changes observed in the crystal structures also occur in solution. While different substrate bound intermediates demonstrate the conformational changes in Dpo4, no significant structural transitions were observed in

the Dpo4 finger domain. The finger domain shows very limited movement from the apo form to the DNA-bound forms and is almost unchanged in the binary/ternary forms. Similarly, the finger domains do not demonstrate significant conformational changes between the apo and ternary complex structures of human DNA polymerase κ (hPolκ),29,30 or between the binary and ternary structures of human DNA polymerase ι (hPolι).28 In contrast, the finger domain of replicative and repair DNA polymerases exhibit significant conformational changes upon nucleotide binding.19,20,38 It is well known that the finger domain contacts the replicating base pair in the active site and is largely responsible for the fidelity-checking mechanisms of nucleotide incorporation. Substrate-induced conformational changes in the finger domains of replicative DNA polymerases increase nucleotide incorporation fidelity. Thus, the preformed active site and the conformational insensitivity of Dpo4 to substrate binding would indicate a lack of base pair checking mechanisms in Dpo4 and possibly all Y-family DNA polymerases. The open active site (with a smaller finger domain and limited contacts with the replicating base pair at the template-primer junction) have been used to explain the error-prone nature and lesion bypass ability of the Y-family polymerases.5 However, the conformational rigidity of the finger domain reported here may also contribute to the low fidelity that characterizes these Y-family DNA polymerases. In contrast to the rigidity of the finger domain, the LF of Dpo4 is flexible regarding its relative positions and orientations with respect to the polymerase as a whole. The LF undergoes a significant conformational change upon DNA binding and is more flexible than the other structural domains of Dpo4 in the DNA-bound structures (Fig. 2d). The flexibility of the LF domain has been observed in other Y-family DNA polymerases, thus possibly indicating a common feature in all Y-family DNA polymerases. For instance, a large conformational change was observed in the LF domain of hPolκ structures in the presence and in the absence of DNA.29,30 This human

326

Snapshots of a Y-Family DNA Polymerase in Replication

Y-family polymerase has the LF domain docked against the polymerase core (the opposite side of the central cleft) and contacts the palm and thumb domain with a 600 Å2 interface in its apo form (Fig. 9c).29 In a fashion similar to that observed here with Dpo4, the LF finger domain of hPolκ undergoes a conformational change and contacts the finger domain when it binds the major groove of the dsDNA in the ternary complex structure.30 An additional interface of 1600 Å2 between Polκ and the dsDNA stabilizes the DNA-bound conformation of hPolκ in which a relatively small interface of 140 Å2 is buried between the LF and finger domains. Conversely, the LF domain in the apo yeast Polη structure8 interacts with the finger domain, generating a ∼1000 Å2 interface, and is thus observed in a position similar to that in the DNA-bound form.39 The large interface results from an additional 37 amino acid residue, three β-strand motif in the finger domain, which is noticeably absent from other Y-polymerases. Further analysis indicates that the flexible nature of the LF domain is still apparent in yPolη. The structures of yPolη in the apo and DNA-bound forms revealed a re-orientation of the LF by about 8°, which is significant but less than the expected rotation.8,39 According to a study of yPolη modeled upon the Dpo4 ternary structure,11 the LF domain of yPolη would require a 45° rotation in order to optimally bind to the DNA major groove. Interestingly, the overall conformations of Y-family DNA polymerases in DNA-bound forms are superimposed well across most available structures in the family with the exception of yPolη.5,28,30,40 The Y-family polymerases bind DNA in the same manner as Dpo4 by using the thumb domain and the LF domain to grasp dsDNA along the minor groove and the major groove, respectively. It seems that the DNA helix serves as a scaffold to keep the overall structures of the Y-family polymerases in an identical conformation once DNA is bound.

The LF domain is required for the proper function of the Y-family polymerases.5,7,9 The structural conservation of the DNA-binding mode implies an important role of the LF domain in the polymerase function. It has been demonstrated that the LF domain of Dpo4 is responsible for interactions with its key cofactor, β-clamp or proliferating cell nuclear antigen.41 Interactions between the Escherichia coli βclamp and the LF domain of E. coli Pol IV, a Y-family DNA polymerase, have been observed in a crystal structure.42 The flexible linker between the LF domain and the polymerase core may make the LF domain more accessible to its co-factor proteins at the replication fork, as well as to provide a wider range of possible domain orientations that may be necessary for proper protein–DNA contacts during nucleotide incorporation. Implications of local conformational changes Previous kinetic studies on the A- and B-family DNA polymerases have identified a common ratelimiting step in nucleotide incorporation, which corresponds to a protein conformational change that is crucial to both catalytic efficiency and polymerase fidelity.17 Pre-steady-state kinetic studies of two Y-family DNA polymerases, Dpo416 and yeast Polη,25 indicate that the Y-family DNA polymerases also utilize an induced-fit mechanism. X-ray crystallographic studies of DNA polymerases in the A-, B-, and X-families have demonstrated significant structural changes in their finger domains.19,20,35,43,44 These significant conformational changes in the finger domain have been proposed to represent the rate-limiting protein conformational change step identified in these aforementioned kinetic studies. However, a recent fluorescence resonance energy transfer study of the A-family DNA polymerase Klentaq1 by Rothwell et al. indicates that the motion of the finger domain is too fast to limit nucleotide

Fig. 9. The apo, unliganded structures of Y-family polymerases. (a) Dpo4 (this work); (b) yPolη (1JIH);8 (c) hPolκ (1T94).29 All the polymerase structures are depicted to the same scale and the same orientation of their palm domains by superposition of all the structures on the conserved palm domains. The polymerases are colored as in Fig. 2: finger domain, blue; palm domain, red; thumb domain, green; and little finger domain, purple. The positions of LF domains (purple) are varied relative to the polymerase cores in three apo Y-polymerases. The LF domains of Dpo4 and hPolκ are dissociated from their finger domains, while the LF domain of yPolη remains in touch with the finger domain.

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327

incorporation.21 Rather, they proposed that a local structural rearrangement may be the rate-limiting step. In parallel, studies on DNA repair polymerase β also concludes that either subtle side chain or DNA conformational changes, but not the finger domain movement, may account for the rate-limiting step in nucleotide incorporation.45,46 Clearly, no open-toclosed conformational transitions has been observed between the binary and the ternary complexes of Yfamily DNA polymerases.5,27,28 With the apo and binary structures solved at 1.9 Å and 2.2 Å resolution, our present work reveals local conformational changes at the nucleotide-binding site of Dpo4 upon nucleotide binding. The observation of different side chain conformations between binary and ternary complexes of Dpo4 have been observed with Dpo4 complexed to lesion-containing DNA structures.31 This supports a general structural model proposed from the studies of Klentaq1 and pol β that the rate-limiting step may be due to local conformational changes in DNA polymerases. The significance of the localized structural changes is supported also by a previous mutagenesis study with yPolη.35 The nucleotide-binding residues of Dpo4 involved in these local conformational changes, specifically Arg51, Tyr48 and Lys159, are highly conserved among Y-family DNA polymerases. Mutation of Tyr64 (equivalent to Tyr48 of Dpo4) of yPolη to phenylalanine dramatically reduces the nucleotide incorporation efficiency of yPolη,35 indicating the importance of the tyrosine hydroxyl group in H-bonding to the triphosphate of dNTP. Furthermore, the overall process involving local conformational changes may also include proper metal ion coordination at the active site. In support of this, we have observed that the triphosphate moiety of a correct nucleotide is required for two metal ion coordination, especially for the metal ion at the A site in this work and in the mis-insertion structures of Dpo4.13 Thus, it is possible that the rate-limiting protein conformational change step:

domain and several local conformational changes at the nucleotide-binding site during nucleotide incorporation. In addition, the rigid nature of the finger domain of Dpo4 limits its ability to distinguish efficiently an incorrect from a correct base pair in the enzyme active site.

E DNA dNTP X EV DNA dNTP

d

d

d

d

in the kinetic mechanism involves the collective movements of specific amino acid side chains and the proper coordination of metal ion binding.

Conclusions Our crystal structures illustrate atomic details of a Y-family polymerase in three different stages of catalysis: the apo-, binary and ternary forms in high resolution. In combination with the results of the solution-based tryptophan fluorescence study, the structural studies provide compelling evidence that conformational changes occur in Dpo4 during the catalytic cycle. Instead of the “open to closed” conformational change induced in high-fidelity DNA polymerases, particularly in the context of the finger domain, Dpo4 undergoes a large-scale conformational change upon DNA binding in the LF

Experimental Procedures Protein and DNA preparation A pair of forward and reverse primers (50-mer) was designed to remove 11 residues from the C-terminus of Dpo4: Forward: 5′-GGAGTAAGGTTCAGTAAATTTATTACTTAAGGATCCGAATTCGAGCTCCG-3′ Reverse: 5′-CGGAGCTCGAATTCGGATCCTTAAGTAATAAATTTACTGAACCTTACTCC-3′). Using the QuikChange site-directed mutagenesis kit (Stratagene), a PCR reaction was performed to create the p1914-Cterm plasmid. DNA sequencing verified the truncated dpo4 gene missing the sequence coding for amino acid residues 342–351. The full-length Dpo4 and its C-terminal truncation mutant were over-expressed and purified as described.47 The purified Dpo4 was concentrated to 18–20 mg/ml in a buffer containing 20 mM Hepes (pH 7.0), 0.1 mM EDTA, 2.5% (v/v) glycerol, and 0.1 M NaCl. The DNA oligonucleotides used for crystallization were purchased from the V. M. Keck facility at Yale University† and purified by HPLC. The complementary primer: 5′-GGGACCCTTCGAAT-3′ and template 5′-TTTTATTCGAAGGGTCCC-3′ were annealed by mixing equal molar amounts of primer and template, and subsequently heating the mixture at 90°C for 5 min, followed by gradual cooling to 4°C. The Dpo4 Y274W mutant used for fluorescence spectroscopy was made by site-directed mutagenesis using the following primers: Forward: 5′-TAGAGCAATAGAAGAATCATATTGGAAGTTAGATAAGAGGATTCC-3′ Reverse: 5′-GGAATCCTCTTATCTAACTTCCAATATGATTCTTCTATTGCTCTA-3′ The mutant protein was purified as described above, with dialysis in a buffer consisting of 25 mM Na3PO4 (pH 7.5), 50 mM NaCl, 5 mM MgCl2, and 5% glycerol. The duplex DNA used for the fluorescence experiments was made by mixing equal molar amounts of an 18mer template strand: 5′-TCAGGCATTCCTTCCCCC-3′ with a 13mer primer: 5′-GGGGGAGGAATG-3′

† http://ww.keck.med.yale.edu/oligoes

328

Snapshots of a Y-Family DNA Polymerase in Replication

Crystallization

Fluorescence spectroscopy

The crystals were grown at room temperature using the hanging drop vapor diffusion method. Truncated Dpo4 in concentration of 10 mg/ml was crystallized with a wellbuffer of 0.2 M lithium nitrate and 20% PEG3350 at room temperature. The Dpo4-DNA binary complex crystals were generated by mixing purified full-length Dpo4 protein with pre-annealed primer/template DNA at a 1:1.2 molar ratio and incubated for 20 min at room temperature. The hanging drop was made by mixing 10 mg/ml protein–DNA complex solution with an equal volume of well buffer containing 0.2 M calcium acetate, 0.1 M Hepes (pH 7.0), 5% glycerol, and 10–15% PEG 3350. Cryo-buffer was well buffer with 20% PEG 3350 and 15% glycerol.

Spectra were obtained using a Fluorolog-3 spectrofluorimeter (Jobin Yvon Inc). Tryptophan fluorescence was determined by exciting at 290 nm and recording the emission from 300–450 nm with a 2 nm bandpass. The annealed primer-template DNA was added to 2 μM Dpo4 Y274W with a final protein/DNA molar ratios of 1:1.5. Primer-template DNA was added by mixing 10 μl of 0.18 mM DNA into 590 μl of the Y274W protein sample. The resulting volume increase was 1.6%.

Data collection and structure determination Data for both the apo- and DNA-bound Dpo4 crystals were collected at the home source using a Rigaku-MSC RU200 generator and a mar345 image plate detector (Table 1). Crystals were soaked for 1 min in cryo-buffers before they were mounted into the liquid nitrogen stream. The cryostream was supplied by the Oxford cryo-cooling system. The apo-Dpo4 crystal diffracted to 1.9 Å while the binary Dpo4 crystal diffracted to 2.2 Å. X-ray diffraction data were processed using DENZO and scaled using SCALEPACK.48 Both structures were solved by molecular replacement (MR) using PHASER,49 with the type I Dpo4 (1JX4) structure used as the search model. In the apo structure determination, the search model was divided into two independent parts: the polymerase core and the LF domain, to accommodate conformational changes. Rebuilding the structures was refined by using REFMAC50 and CNS.51 The structural models were built and adjusted using graphics program O and COOT.52,53 An anomalous difference map was generated for the binary Dpo4-DNA structure in order to identify the positions of calcium atoms unambiguously. Interface area calculations were performed with PISA‡.54 The structure Figs. 2–7 and 9 were generated using PYMOL§. Degradation of primer/template by Dpo4 A solution of Dpo4 (240 nM) was incubated with one of two 22/41-mer DNA substrates (60 nM): 22-mer: 5′-CGCAGCCGTCCACCAACTCAA-3′ containing either a terminal mismatch: 41-mer: 5′-GGACGGCATTGGATCGACGATGAGTTGGTTGGACGGCTGCG-3′) or a matched terminus: 41-mer: 5′-GGACGGCATTGGATCGACGTTGAGTTGGTTGGACGGCTGCG-3′ for various reaction times before being quenched with 0.37 M EDTA. The reaction mixtures were analyzed by sequencing gel electrophoresis. The products were quantified using PhosphoImager 445 SI (Molecular Dynamics). The plots of product concentration versus reaction time were fit to the following equation: ½Product ¼ A expðkexotÞ

‡ http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html § http://pymol.sourceforge.net/

Protein Data Bank accession codes The atomic coordinates and structural factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession codes 2RDI for apo-Dpo4 and 2RDJ for DNAbound Dpo4.

Acknowledgements This work was supported by the Canadian Institutes of Health Research grant MOP-67128 (to H.L.). Z.S. was supported by the National Science Foundation Career Award MCB-0447899. The fluorescence experiments were carried out at the Biomolecular Interactions and Conformations Facility at the Schulich School of Medicine and Dentistry of the University of Western Ontario; the assistance of the Facility Manager, Lee-Ann Briere, is gratefully acknowledged. We thank Dr Stanley Dunn for critical reading of the manuscript.

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