The objectives of this work were to investigate the conformational changes that occur when a dNTP is inserted opposite an oxoG template base in the active site of Dpo4, and the subsequent incorporation of this nucleobase into the nascent DNA strand. Since translesion bypass of oxidatively damaged DNA templates by Dpo4 has not been previously investigated to our knowledge, we first surveyed the kinetics of translesion bypass of oxoG in order to select the most appropriate dNTP to be inserted opposite this lesion in our subsequent co-crystal structural studies.
Fidelity of Translesion Synthesis and Primer Extension Kinetics
The results of steady-state, one-step primer extension experiments are summarized in . Primer extension studies in vitro utilizing all four dNTPs show that Dpo4 readily elongates past guanine (G) or oxoG lesions in the template strand to produce full-length, 19-mer extension products (A). Michaelis-Menten parameters Vmax and Km (), were determined for the dNTP insertion step opposite oxoG (or G at the same site) (B), and for the one-step extension step beyond an oxoG•C or an oxoG•A base pair (C). The insertion frequency, fins, of dCTP opposite oxoG is only 2-fold smaller than it is opposite G, with the fins values opposite oxoG decreasing in the order dCTP > dATP, dTTP > dGTP (). Interestingly, the fext value is significantly greater for the one-step extension beyond an oxoG•C base pair than beyond a normal G•C base pair at the same site. Also, extension beyond an oxoG•A mispair is ~30 times smaller than extension from an oxoG•C base pair (). Taken together, these kinetic primer extension data suggest that the oxoG lesion is readily bypassed by Dpo4 in a predominantly error-free manner. In view of our primer extension results catalyzed by Dpo4, we have focused our structural studies on complexes of oxoG-modified template-primer DNA and Dpo4, in the absence and presence of incoming dCTP as well as covalently incorporated C opposite the oxoG lesion site.
Kinetic Parameters of Insertion and Extension Catalyzed by Dpo4
Fidelity and Efficiency of Base Incorporation on Unmodified G and oxoG-Modified Templates by Dpo4
DNA Substrate Design and Crystal Structure Determination
In order to capture Dpo4 conformational changes during DNA synthesis, we crystallized this bypass polymerase in three types of complexes: the preinsertion binary complex containing oxoG (or G) 19-mer template and 13-mer primer strand (A), the insertion ternary complex with a dCTP at the active site paired with oxoG (or G) in the template strand (B), and the postinsertion binary complex with the incorporated C residue at the 3′ end of the primer paired with oxoG in the template strand (C). All three types of complexes () belong to the P21 space group with distinct unit cell parameters and with two complexes per asymmetric unit ().
Structures and Intermolecular Contact Details of Dpo4 Complexes with oxoG-Modified Template-Primer DNA
Crystal Data, Data Collection, and Refinement Statistics
The structure of the Dpo4 oxoG-modified insertion ternary complex was solved by molecular replacement employing the published Dpo4 ternary complex with an 18-mer unmodified template–12-mer primer junction and incoming ddADP [5
] as a search model, and refined to 1.95-Å resolution. The molecular replacement method was employed to solve the structures of the preinsertion and postinsertion binary complexes using the oxoG-modified insertion ternary complex as a search model. The crystal data, together with the data collection and refinement statistics for all structures, are summarized in .
Structure of the oxoG-Modified Preinsertion Binary Complex
We have determined the crystal structure of the Dpo4 preinsertion binary complex with oxoG-modified 19-mer template/dideoxy-terminated, 13-mer primer at 2.35-Å resolution (Figure S1
A). The corresponding structure of the preinsertion binary complex with unmodified G was solved at 2.7-Å resolution (Figure S2
A). The 5′-C-T-A-A-C-oxoG (or G) single-stranded template overhang is disordered in both crystals. The structures of the oxoG-modified and unmodified preinsertion binary complexes are similar, with root mean square deviation (RMSD)
= 0.68 Å; therefore we focus on the description of the oxoG-modified complex. The alignment of residues in the Dpo4 polymerase active site pocket is outlined in D, with the C7•G13 base pair (the numbering scheme is defined in A), adjacent to the oxoG lesion, enclosed in a box for reference. The roof of the active site, formed by the finger domain (in blue), is positioned directly over this C•G pair in the preinsertion binary complex (The Dpo4 bears only one Ca2+
cation, coordinated by invariant D7, D105, and E106 residues D).
The Dpo4 polymerase embraces the 19-mer template/13-mer primer DNA by its four domains: palm (residues 1–10 and 78–166), finger (residues 11–77), thumb (residues 167–233), and little-finger (residues 244–341) (see Figures S1
A and S2
A). The thumb is joined to the little-finger domain by a 10-amino-acid-long tether (residues 234–243) that allows positioning of the little finger on the other side of the DNA duplex. The contacts between Dpo4 amino acid residues and the DNA in the oxoG-modified preinsertion binary complex (see Figure S1
A) are depicted in G. The phosphate groups interacting with the thumb domain within the minor groove are shown in red, and the phosphate groups interacting with the little-finger domain within the major groove are shown in purple, with the conserved interactions boxed for both domains.
Structure of oxoG-Modified Insertion Ternary Complex with Incoming dCTP
We have determined the 1.95-Å crystal structure of the Dpo4 insertion ternary complex with oxoG-modified, 19-mer template/dideoxy-terminated 13-mer primer, and dCTP positioned opposite oxoG (see Figure S1
B). The corresponding structure of the insertion ternary complex with unmodified G was solved at 2.80-Å resolution (Figure S2
B). Residues 3–19 of the template strand in the oxoG-modified complex and the entire template strand in the unmodified complex were successfully traced in the electron density map. The structures of the oxoG-containing and unmodified insertion ternary complexes are similar with RMSD
= 0.60 Å; therefore we focus on the description of the oxoG-modified complex.
The finger domain, which is on top of the C7•G13 base pair in the active site of the preinsertion binary complex (D), is now positioned over the replicating oxoG(anti)
•dCTP base pair (A) in the active site of the insertion ternary complex (see E). The side chains of the hydrophobic residues V32 and A42 and the backbone of G58 are packed against the purine ring of oxoG, and the hydrophobic residues A44, A57, and Y12 are packed against the pyrimidine and sugar rings of incoming dCTP. The positioning of the triphosphate backbone of dCTP within the catalytic pocket is shown in B. Details of the intermolecular contacts between Dpo4 and the oxoG-containing template-primer junction and the dCTP in the insertion ternary complex (see Figure S1
B) are shown schematically in H.
Comparison of Pairing Alignments and Intermolecular Interactions Involving oxoG-Modified and Unmodified G Residues in Insertion Ternary Complexes with Incoming dCTP
Four divalent cation sites were identified in the first molecule, and three sites were found in the second molecule within the asymmetric unit of the oxoG-modified insertion ternary complex. Since the crystallization conditions for the Dpo4-DNA complexes included 100 mM calcium and 5 mM magnesium ions, we used the anomalous signal of the calcium ion at 1.5418 Å (Cu Kα radiation) to distinguish it from the much weaker anomalous scattering by the magnesium ion at this wavelength. Seven strongest peaks on the anomalous map allowed us to assign all ions to calcium. We find one Ca2+ ion coordinated by the catalytic triad, the second chelated by the phosphate groups of the incoming dCTP, with the third ion coordinated by the loop of the thumb domain (residues 181 and 186), adjacent to the tip of helix H (C). The fourth calcium ion (absent in the second molecule of the asymmetric unit) is bound between adjacent C•G and A•T base pairs in the free portion of the double-stranded DNA region.
Comparison of oxoG versus G Alignments in Insertion Ternary Complexes
•dCTP (A) and G(anti)
•dCTP pairing alignments are of the Watson-Crick type in their respective insertion ternary complexes. The sugar-phosphate backbone of the oxoG and G residues and their interactions with amino-acid side chains in their respective insertion ternary complexes are shown in D and E, respectively. Accommodation of the oxoG carbonyl functionality within the Dpo4 polymerase active site results in a shift of the phosphate group of the oxoG base by 3.5 Å and a change in the backbone torsion angle α (O3′-P-O5′-C5′) by 176° as compared with the unmodified structure (superimposed segments compared in F), with a similar observation reported previously for the pol β complex [33
]. The oxygen atoms of the relocated phosphate group form two hydrogen bonds with the guanidine group of Arg331, one hydrogen bond with the guanidine group of Arg332, and two hydrogen bonds with the hydroxyl group of Ser34 (D). The carbonyl oxygen at C8 forms a water-mediated hydrogen bond with the side chain of Arg332, thus helping to lock the oxoG residue in the anti
conformation (D). By contrast, the phosphate group of unmodified G forms only two hydrogen bonds with the guanidine group of Arg332 (E); thus oxoG(anti)
forms four more hydrogen bonds with Dpo4 (D) than unmodified G does (E).
The dCTP-Binding Step: Conformational Transitions from Preinsertion Binary to Insertion Ternary Complex
We now compare the structures of the preinsertion binary complex with the insertion ternary complex that contains a dCTP molecule opposite oxoG by superpositioning their DNA duplexes in order to identify conformational changes associated with translocation of the polymerase during the dCTP-binding step. This superposition for the entire complex is shown in A and B, with little-finger domain–DNA interactions shown in C and thumb domain–DNA interactions shown in D. The key observation is that the finger, palm, and little-finger domains move up by one nucleotide to allow for the noncovalent insertion of dCTP into the active site (C, with details shown in ), while the thumb maintains its contacts with the DNA duplex (shown by arrows in D, with details shown in ).
Conformational Transitions of Dpo4 and oxoG-Modified Template-Primer DNA Associated with the Nucleotide-Binding Step Following Superimposition of DNA Duplexes
Details of Changes in Dpo4 Little-Finger Domain Contacts with Backbone Phosphates on the oxoG-Modified Template-Primer DNA Associated with the Nucleotide Binding and Incorporation Steps
Details of Changes in Dpo4 Thumb Domain Contacts with Backbone Phosphates on the oxoG-Modified Template-Primer DNA Associated with the Nucleotide Binding and Incorporation Steps
The little finger slides and rotates counterclockwise (with respect to the 5′-to-3′ direction of the template strand) as a rigid entity along the DNA duplex, resulting in translocation of the polymerase by one step (C), thereby allowing the next template base and incoming dCTP to enter the active site. Upon dCTP binding, the contacts of the little finger with the phosphate groups of residues T8, A9, C10, and C11 (A) are relocated to the phosphate groups of C7, T8, A9, and C10 (C) on the template strand. At the same time, related little-finger contacts on the primer strand are displaced from the phosphate groups of G5, G6, and A7 (B) to the phosphate groups of G6, A7, and T8 (D). It is interesting that the contacts between the DNA phosphate groups and the little finger, which is unique among Y-family bypass polymerases, involve electrostatic interactions between alternating arginine and peptide backbone nitrogen groups (A and C). The little finger provides an interface along which the DNA can more easily slide during the translocation step in a manner that is independent of base sequence (see C). The little finger is positioned closer to the primer strand in the preinsertion binary complex than in the insertion ternary complex, thus allowing for additional hydrogen-bonding contacts with the phosphate groups of T8 in the template strand (A) and G5 in the primer strand (B) in the former complex. By contrast, as a result of the dCTP binding, the α-helical segments of the thumb domain (helixes H, K, J) change their relative position with respect to the palm domain, and even though they rotate counterclockwise relative to the DNA, they maintain the same contacts with the phosphate of T13 of the template strand (A and C) and with the backbone phosphates of T11 and A12 of the primer strand (B and D).
In quantitative terms, dCTP binding results in rotation of the little finger with respect to the DNA by 29°, almost its full-twist value, while the palm and finger domains, as well as the thumb domain, rotate by 18°, half of their full rotation cycle (). The little-finger domain, as well as the palm and finger domains, moves along the DNA axis by 3.2–3.5 Å, generating space for nascent base-pair formation. By contrast, the thumb domain, which maintains its contacts with DNA on bonding of dCTP, does not translate with respect to the DNA ().
Rotation and Translation of Dpo4 Domains with Respect to DNA for the Preinsertion Binary to Insertion Ternary Step (dCTP Binding) and for the Insertion Ternary to Postinsertion Binary Step (dCTP Incorporation)
Structure of the oxoG-Modified Postinsertion Binary Complex with Covalently Incorporated Cytosine Opposite oxoG
In the case of the postinsertion binary complex, a covalently incorporated cytosine is positioned opposite the oxoG site (see C) and forms a Watson-Crick oxoG•C pair. Residues 3–19 of the template strand can be traced in the electron density map. The finger domain is positioned directly over the Watson-Crick oxoG•C pair with one Ca2+
cation coordinated by the D7, D105, and E106 at the active site of Dpo4 (see F). Details of the intermolecular contacts between Dpo4 and the oxoG-containing template-primer junction in the postinsertion binary complex (Figure S1
C) are shown schematically in I.
The Cytosine-Incorporation Step: Conformational Transitions from Insertion Ternary Complex to Postinsertion Binary Complex
We compare the structures of the insertion ternary complex that contains the incoming dCTP molecule opposite oxoG with the postinsertion binary complex that contains covalently incorporated C opposite oxoG by superpositioning their DNA duplexes in order to identify the conformational changes associated with translocation of the polymerase during the dCTP incorporation step. This superposition for the entire complex is shown in A and B, with little-finger-domain–DNA interactions shown in C and thumb domain–DNA interactions in D. The key observation is that the little-finger domain maintains its contacts with the DNA (C), while the thumb domain moves by one nucleotide (shown by arrows in D) during covalent incorporation of the cytosine opposite the oxoG lesion site. These conformational changes in the thumb domain on cytosine incorporation are accompanied by the release of the pyrophosphate together with a divalent cation.
Conformational Transitions of Dpo4 and oxoG-Modified Template-Primer DNA Associated with the Covalent Incorporation Step Following Superimposition of DNA Duplexes
Upon covalent cytosine incorporation, there is retention of little-finger contacts with the phosphate groups of C7, T8, A9, and C10 on the template strand (see C and E), and the phosphate groups of G6, A7, and T8 on the primer strand (see D and F). By contrast, the contact of the thumb domain with the phosphate group of T13 (see C) is relocated to the phosphate group of A12 (see E) on the template strand. At the same time, related thumb-domain contacts on the primer strand are displaced from the phosphate groups of T11 and A12 (see D) to the phosphate groups of A12 and G13 (see F).
In quantitative terms, covalent cytosine incorporation results in rotation of the thumb domain and palm and finger domains by 17 to 18 degrees, half of their full rotation cycle, while the little finger undergoes a minimal rotation of 3° (). The thumb domain moves along the DNA axis by 2.7 Å, while the little-finger domain, which maintains its contacts with DNA on cytosine incorporation, and palm and finger domains, undergo minimal translation (0.5 Å) with respect to the DNA ().
dCTP Positioning in Insertion Ternary Complex
The position of the substrate, dCTP, with respect to the 3′-end nucleotide, G13, on the primer strand, differs substantially from a B-DNA step, with a reduction in the twist angle and displacement of the substrate base toward the DNA major groove (A). The view looking down the helix axis clearly demonstrates this shifted alignment of dCTP relative to the 3′ portion of the primer strand (B), with the sugar ring of dCTP positioned significantly closer to the sugar ring of G13, when compared with the typical B-DNA separation between A12 and G13. Upon covalent base incorporation, the restoration of the B-DNA conformation between the nascent base pair and its adjacent base pair (C) propagates toward the 5′ end of the primer strand. When viewed down the helix axis, the separations between the sugar rings of G13 and the newly incorporated base, C14, and between G13 and A12, are approximately equal (D).
Relative Positioning of the oxoG•dCTP Pair in the Insertion Ternary Complex and the oxoG•C Pair in the Postinsertion Binary Complex
The movement of primer strand 3′-terminal residue with respect to Dpo4 palm and finger domains is defined by the restraining function of the aromatic ring of Y12, which is packed against the sugar ring of the dCTP in the insertion ternary complex and against the sugar ring of newly incorporated C14 base in the postinsertion binary complex (E). The conformational transition accompanying a restoration of the B-DNA step results in the contacts between the α-helical segments of the thumb domain and DNA phosphate backbone shifting by one step.
Base-Pair Progression during dCTP Binding and Cytosine-Incorporation Steps
The DNA molecule retains a B-like conformation in the preinsertion binary, insertion ternary, and postinsertion binary complexes. However, there is a narrowing of the minor groove at the contact sites with the thumb domain and a widening of the major groove at the contact sites with the little finger, which is observed in all complexes, and which increases in the insertion ternary complex relative to both preinsertion binary and postinsertion binary complexes. The C7•G13 pair is adjacent to the oxoG lesion site, and its positioning within/adjacent to the Dpo4 active site is of interest in the three complexes.
The preinsertion binary and insertion ternary complexes associated with dCTP binding can be compared by superpositioning their finger and palm domains, which constitute the Dpo4 active site (A). The positioning of the C7•G13 pair relative to the active site for the two complexes is shown in A. The template strand C7 of the preinsertion binary complex (in silver) is rotated and displaced along the helical axis to a new position in the insertion ternary complex (in color), while primer strand G13 is only displaced to maintain Watson-Crick hydrogen bonding, but is minimally rotated upon dCTP binding.
Conformational Transitions of Dpo4 and oxoG-Modified Template-Primer DNA Associated with Nucleotide Binding and Covalent Incorporation Steps Following Superimposition of Dpo4 Palm and Finger Domains
Comparison of the Relative Progression (Twist Angle Viewed down the Helix Axis) of C7•G13 Pairs between Complexes Superpositioned on their Finger and Palm Domains
The insertion ternary and postinsertion binary complexes associated with dCTP incorporation can be compared by superpositioning their Dpo4 finger and palm domains (see B). The relative positioning of the C7•G13 pair for the two complexes is shown in B. Primer strand G13 of the insertion ternary complex (in color) is rotated and displaced to a new position in the postinsertion binary complex (in beige), while template strand C7 is only minimally displaced and rotated to maintain Watson-Crick hydrogen bonding.
Superposition of the preinsertion binary with the postinsertion binary complexes by their Dpo4 palm and finger domains (Figure S3
A) results in almost ideal overlap between the DNA duplexes, with rotation and displacement with respect to the DNA helical axis by one full nucleotide step (Figure S3
B). This can be best visualized by viewing the positioning of the C7•G13 pairs of the preinsertion binary (in silver) and postinsertion binary (in beige) complexes (C).