DNA polymerase beta substrate specificity: side chain modulation of the "A-rule"

Abstract

Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) beta, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol beta insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol beta bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.

FIGURE 1.

Chemical comparison of analogs for the abasic site and dATP. A, natural abasic site is in equilibrium between the closed sugar ring hemiacetal (left) and the open aldehyde (right) forms. The synthetic closed sugar ring abasic site analog tetrahydrofuran is illustrated below. B, comparison of the chemical structure of adenine and 5-nitro-indolyl.

FIGURE 2.

Relative nucleoside triphosphate insertion efficiency opposite a synthetic abasic site. Relative A specificity was calculated from the ratio of catalytic efficiencies (dNTP/dATP) tabulated in Tables 1 and 2. A, data for Mg²⁺ are illustrated for 22 °C (open bars) and 37 °C (filled bars). Specificity for 5-NITP was not determined at 22 °C. B, data at 22 °C are illustrated for Mg²⁺ (open bars) and Mn²⁺ (filled bars).

FIGURE 3.

Arg-283 modulation of the A-rule. The catalytic efficiency for the preferential insertion of dATP (filled bars) relative to dGTP (open bars) is lost with R283A. This loss in specificity is completely due to the loss in dATP insertion efficiency with the R283A mutant because dGTP insertion was hardly affected. Steady-state kinetic parameters were determined as outlined under “Experimental Procedures” at 37 °C. Catalytic efficiencies are from Table 1.

FIGURE 4.

Closed conformation of the ternary substrate complex with a dATP analog opposite an abasic site. The superimposed binary (abasic site gapped DNA; PDB code 3ISC) and ternary substrate (+dAMPCPP; PDB code 3ISD) complex structures indicate that pol β is in a globally closed conformation. The L-domain (i.e. lyase) and N-subdomain of the ternary complex (purple) move toward the incoming nucleotide relative to their positions in the binary complex (blue and yellow, respectively). The DNA is omitted for clarity. The position of the C- and D-subdomains (gray) is not altered upon nucleotide binding.

FIGURE 5.

Comparison of templated and nontemplated ternary substrate complex structures. A, active site comparison between superimposed ternary complex structures with an active site mismatch (template dG, PDB code 3C2M; light blue carbons) (17) and nontemplating abasic site (PDB code 3ISD, magenta carbons). In both instances, the template residue (n) is shifted upstream ∼3 Å vacating the templating pocket. The incoming dAMPCPP and Mn²⁺ in these structures superimpose well. In contrast, the 3′-OH (O3′) of the primer terminus is better positioned in the structure with the abasic site than in the case of a nascent mismatch. Likewise, Arg-283 (R283) is observed in a new conformation not observed previously. This side chain occupies the template base binding pocket vacated in the mismatch structure but lies under the template backbone in the absence of a template base. The solid arrow indicates the upstream direction from the active site, and the dashed arrow indicates the downstream direction. B, active site comparison between superimposed ternary complex structures with a correct active site base pair (dA-dUMPNPP, PDB code 2FMS; gray carbons) (16) and nontemplating abasic site (magenta carbons). The template THF (magenta, n) is moved upstream of the active site and positioned approximately where the n − 1 (template nucleotide opposite the primer terminus) nucleotide is found with a correct nascent base pair. In this situation, Arg-283 interacts with the minor groove edge of the templating base. The polymerase domain is in a closed conformation as judged by the position of αN.

FIGURE 6.

Alternate side chain conformations for key residues involved in active site signaling. A, Arg-283 is observed in the ternary complex structure in a conformation not observed previously (PDB code 3ISD). In the absence of a templating base (n), the arginine side chain points away from the incoming dAMPCPP and makes a single hydrogen bond with the templating phosphate backbone (dotted green line). The immediate upstream and downstream templating nucleotides are illustrated (n − 1 and n + 1, respectively). Furthermore, the upstream and downstream directions of the template strand are indicated with a solid and dashed arrow, respectively. An F_o − F_c simulated annealing electron density omit map (blue) contoured at 2.0σ showing electron density corresponding to dAMPCPP, the synthetic abasic site, and Arg-283 is shown. B, F_o − F_c simulated annealing electron density omit map (blue) contoured at 3.0σ showing electron density corresponding to Phe-272 in the ternary substrate complex with a synthetic abasic site (magenta carbons). Tyr-271 is also shown. Phe-272 was modeled in two conformations (C1 and C2 with 50% occupancy). These residues are compared with a superimposed binary (yellow) (PDB code 3ISB) and ternary substrate complex with a correct base pair (gray; PDB IB 2FMS) (16). Thus, Tyr-271 is observed in an intermediate position between the inactive binary complex and active ternary complex, whereas Phe-272 appears to equilibrate between the two positions.

FIGURE 7.

Comparison of templated dATP misinsertion and nontemplated insertion. The efficiency of dATP insertion opposite a synthetic abasic site (THF) is similar to that for the misinsertion opposite dC and dG with Mg²⁺ (open bars) and Mn²⁺ (filled bars). In contrast, a templating dA discourages dATP misinsertion relative to an abasic site ∼30-fold. The misinsertion data were taken from Beard et al. (32) and Batra et al. (17) for Mg²⁺ and Mn²⁺, respectively.