William A. Beard

HUMAN DNA POLYMERASE BETA DEOXYRIBOSE PHOSPHATE LYASE : SUBSTRATE SPECIFICITY AND CATALYTIC MECHANISM

DNA polymerase β (β-pol) cleaves the sugar-phosphate bond 3′ to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5′-incised by AP endonuclease, resulting in a 5′ 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of β-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of β-pol are similar. However, k cat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5′ dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of β-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5′-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.

Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase β

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase β are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5′-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J.(1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T•dGTP and A•dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase β and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.

Substrate Binding by Human Apurinic/Apyrimidinic Endonuclease Indicates a Briggs-Haldane Mechanism

Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5′ to a DNA abasic site. We have characterized this reaction by steady-state and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli. The substrate was a 49-base pair oligonucleotide with an abasic site at position 21. This substrate was generated by treating a 49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase. The enzymatic products of the AP endo nicking reaction were a 20-mer with a hydroxyl group at the 3′-terminus and a 28-mer with a phosphodeoxyribose at the 5′-terminus. To obtain maximal enzymatic activity, it was necessary to stabilize the abasic site during treatment with uracil-DNA glycosylase with a reducing agent. Otherwise, a 20-mer with phosphoribose at the 3′-terminus resulted from β-elimination. In agreement with others, Km and kcat were 100 nM and 10 s−1, respectively. Heat treatment of the abasic site-containing 49-mer without enzyme also resulted in conversion to the β-elimination product. The resultant heat degradation product was an efficient inhibitor of AP endo with a Ki of 30 nM. The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+. Electrophoretic mobility shift assays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations. The association and dissociation rate constants for substrate binding to AP endo were determined by using a challenge assay to follow AP endo-substrate complex formation. Heat degradation product together with heparin served as an effective trap for free enzyme. The results are consistent with a Briggs-Haldane mechanism where kon and koff are 5 × 107 M−1 s−1 and 0.04 s−1, respectively (Kd = 0.8 nM), kcat is 10 s−1, and product release is very rapid (i.e. koff,product ≫ 10 s−1). This scheme is in excellent agreement with the measured steady-state kinetic parameters.

Functional Analysis of the Amino-terminal 8-kDa Domain of DNA Polymerase β as Revealed by Site-directed Mutagenesis DNA BINDING AND 5′-DEOXYRIBOSE PHOSPHATE LYASE ACTIVITIES

The amino-terminal 8-kDa domain of DNA polymerase β functions in binding single-stranded DNA (ssDNA), recognition of a 5′-phosphate in gapped DNA structures, and as a 5′-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed inEscherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)16. Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5′-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5′ terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys35 is involved in 5′-phosphate recognition of DNA polymerase β. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA. Alanine mutants of Lys35 and Lys60 are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys72 resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine mutants of Lys68 and Lys84 had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys72 and Lys35 were additive. The results suggest that Lys72 is directly involved in formation of a covalent imino intermediate and are consistent with Lys72as the predominant Schiff base nucleophile in the dRP lyase β-elimination catalytic reaction.

Role of the "Helix Clamp" in HIV-1 Reverse Transcriptase Catalytic Cycling as Revealed by Alanine-scanning Mutagenesis

Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the “helix clamp” (Hermann, T., Meier, T., Götte, M., and Heumann, H.(1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two α-helices (αH and αI) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in αH make important interactions with nucleic acid and influence frameshift fidelity. To define the role of αI (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of αH, alanine substitution in αI did not have a significant effect on template•primer (T•P) binding as revealed by a lack of an effect on Km,T−P, Ki for 3′-azido-2′,3′-dideoxythymidine 5′-triphosphate, koff,T−P, and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of αI lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T•P (L282A and R284A) demonstrated that these mutant enzymes could bind T•P stoichiometrically and tightly. In contrast, active site concentrations determined from “burst” experiments suggest that the lower activity is due to a smaller population of enzyme bound productively to T•P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of αH and the DNA minor groove, 3-5 nucleotides from the 3′-primer terminus, are suggested to be critical for DNA binding and fidelity.

Probing Structure/Function Relationships of HIV-1 Reverse Transcriptase with Styrene Oxide N2-Guanine Adducts

Details of the interactions between the human immunodeficiency virus (HIV-1) reverse transcriptase and substrate DNA were probed both by introducing site-specific and stereospecific modifications into DNA and by altering the structure of potential critical residues in the polymerase. Unadducted 11-mer DNAs and 11-mer DNAs containing R and S enantiomers of styrene oxide at N2-guanine were ligated with two additional oligonucleotides to create 63-mers that served as templates for HIV-1 reverse transcriptase replication. Oligonucleotides that primed synthesis 5 bases 3′ to the adducts could be extended up to 1 base 3′ and opposite the lesion. However, when the positions of the 3′-OH of the priming oligonucleotides were placed 1, 2, 3, 4, 5, and 6 bases downstream of the styrene oxide guanine adducts, replication was initiated, only to be blocked after incorporating 4, 5, 6, and 7 bases beyond the lesion. The sites of this adduct-induced termination corresponded to the position of the DNA where α-helix H makes contact with the DNA minor groove, 3-5 bases upstream of the growing 3′ end. In addition, mutants of the polymerase in α-helix H (W266A and G262A) alter the termination probabilities caused by these DNA adducts, suggesting that α-helix H is a sensitive monitor of modifications in the minor groove of newly synthesized template-primer DNA several bases distal to the 3′-OH.