3′-Azido-3′-deoxythymidine-resistant Mutants of DNA Polymerase β Identified by in Vivo Selection

Abstract

We developed an in vivo selection to identify 3-azido-3-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase ␤ (pol ␤). The selection utilizes pol ␤'s ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol ␤ allows SC18-12 cells to grow, but they depend on pol ␤ activity, so inhibition of pol ␤ by AZT kills them. We screened a library of randomly mutated pol ␤ cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants. We purified two enzymes with nonconservative mutations in the palm domain of the polymerase. The substitutions D246V and R253M result in reductions in the steadystate catalytic efficiency (K cat /K m ) of AZT-TP incorporation. The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance. The R253M enzyme exhibits significantly higher K m (dTTP) and K cat (dTTP) values, implying that the incorporation reaction is altered. These are the first pol ␤ mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.Efficient and accurate synthesis of DNA, during replication and repair, is essential to the integrity of any genome. Template-directed synthesis requires that a polymerase select the appropriate deoxynucleotide triphosphate (dNTP) and exclude incorrect bases. The interaction between a polymerase and substrates must therefore be highly specific, yet flexible, in order to maintain sequence fidelity. The smallest of the eukaryotic enzymes that accomplish this reaction is DNA polymerase ␤ (pol ␤), 1 a mammalian polymerase which fills short gaps in DNA (1). pol ␤ has been implicated in base excision repair (2, 3) and meiosis (4). pol ␤ is highly suitable for structure-function studies of the molecular mechanism of DNA synthesis due to the availability of information on the polymerase-DNA-substrate ternary complex (5, 6).DNA synthesis by pol ␤ can be compromised by the nucleoside analog drug AZT, which closely resembles a normal substrate. AZT-triphosphate (AZT-TP) presents a normal thymine moiety which may form a Watson-Crick base pair with adenosine, but has a modified sugar ring that results in chain termination. pol ␤ incorporates AZT into DNA in vitro (7) implying that pol ␤ is not able to distinguish perfectly between the drug and the natural nucleotide substrate. This susceptibility of pol ␤ makes AZT resistance a useful probe of enzyme-substrate interactions involved in DNA synthesis.The molecular basis for polymerase substrate specificity, and specifically AZT discrimination, is not well understood. The clinical problem of AZT-resistant HIV has been attributed to mutations in HIV reverse transcriptase. However, the mutant reverse transcriptase enzymes have exhibited little or no change in AZT-TP incorporation...

The E249K Mutator Mutant of DNA Polymerase β Extends Mispaired Termini

“…AZT Incorporation Assay-The ability of each enzyme to incorporate dTTP and AZT-TP into a primer-template was tested in steady-state incorporation reactions using annealed 16-and 45-mer as described previously (16). Reactions were conducted at 37°C in 50 mM Tris, pH 8.0, 10 mM MgCl 2 , 20 mM NaCl, 2 mM DTT, 0.2 mg/ml BSA, 2.5% glycerol, and 200 nM 32 P end-labeled primer-template.…”

“…Expression and Purification of Mutant Enzymes-WT and mutant proteins were expressed from the pHis-␤ vector, which expresses pol ␤ as a fusion protein with a six-residue poly-histidine tag at the N terminus. The enzymes were purified as described previously, using Ni 2ϩ -charged His-bind resin from Novagen or Ni 2ϩ -nitrilotriacetic acid resin from Qiagen (16).…”

“…In this system, pol ␤ substitutes for DNA polymerase I in DNA replication at nonpermissive temperature. The AZT-resistant mutants were selected from a randomly mutagenized library of pol ␤ clones for their ability to grow on AZT in the recA718 polA12 strain at nonpermissive temperature (16). The first two AZT-resistant mutants we described, R253M and D246V, carried nonconservative substitutions in the palm domain (16).…”

“…The AZT-resistant mutants were selected from a randomly mutagenized library of pol ␤ clones for their ability to grow on AZT in the recA718 polA12 strain at nonpermissive temperature (16). The first two AZT-resistant mutants we described, R253M and D246V, carried nonconservative substitutions in the palm domain (16). The strong phenotypes exhibited in vivo and in vitro by the R253M and D246V mutants called attention to amino acid residues 240 -253 that comprise a loop located within the palm domain of pol ␤, in which three different AZT resistance mutations were identified.…”

A DNA polymerase β mutant from colon cancer cells induces mutations

“…The DNA substrates used in the biochemical assays described below are displayed in Table 1. The primer oligonucleotides were gel-purified as described (31) and were radiolabeled at the 5Ј end by standard methods by using T4 polynucleotide kinase (New England BioLabs) and [␥-32 P]ATP. The oligonucleotides 45A-22-22, CII45-CIIU-CIID, and CII45T-CIIU-CIIDA were annealed as described (20).…”