Search

We have used random sequence mutagenesis and complementation in a bacterial selection system to establish a large library of immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with amino acid substitutions in the beta3-beta4 region of the fingers subdomain [Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) J. Biol. Chem. 271, 4872-4878]. We show here that one of these mutants, D76V, exhibits increased accuracy of copying both DNA and RNA templates in a primer extension assay with biased dNTP pools. More detailed analysis of DNA-dependent polymerization showed that the D76V mutation conferred an up to 14-fold increase in fidelity of nucleotide insertion and a 9-fold reduced mutation rate in an M13mp2 lacZalpha forward mutation assay. Substitution at D76 with positively charged (D76R) and nonpolar (D76V and D76I) residues increased replicational accuracy, while substitutions with negatively charged (D76E) and polar residues (D76S and D76C) had little effect on fidelity. We propose that D76 affects replicational accuracy by mediating interaction between the fingers subdomain and the single-stranded template. Our work shows that the Escherichia coli complementation system can yield HIV RT mutants with increased fidelity that have not been isolated from the natural host and that are valuable in understanding the molecular bases of replicational accuracy.

Reactive oxygen species, produced in cells by a variety of mechanisms, damage DNA and cause mutations. To characterize the types of mutations produced in mammalian cells, we copied DNA damaged by reactive oxygen species with mammalian DNA polymerase beta. Double-stranded circular M13mp2 DNA containing a 361-nucleotide single-stranded gap within the lacZ gene was damaged by aerobic incubation with Fe2+ and H2O2. The gap then was filled by purified recombinant rat DNA polymerase beta, and the DNA was transfected into Escherichia coli. Mutations within the nonessential lacZ gene for beta-galactosidase were identified by reduced alpha-complementation. In this system, oxidative damage increased the mutation frequency within the target region by an average of 4.3-fold. At certain sites, the base substitution rate is nearly 300 times greater than would be expected to result from a random distribution of damage. The oxidatively induced mutations fall into two categories: those apparently caused by direct miscoding of modified DNA and those associated with enhanced misincorporation at prexisting polymerase-specific hot spots. The latter group may be due to a conformational change in the DNA caused by oxidative modification and could be indicative of a novel mutagenic mechanism.

DNA polymerase III of the yeast Saccharomyces cerevisiae has been reported to be encoded at the CDC2 locus based on two observations. First, the CDC2 gene has homology to known DNA polymerase genes [Boulet et al. (1989) EMBO J. 8, 1849-1854], and second, the mutants cdc2-1 and cdc2-2 yield little or no DNA polymerase III activity in vitro [Boulet et al. (1989); Sitney et al. (1989) Cell 56, 599-605]. We describe here the isolation of temperature-sensitive DNA polymerase III from cdc2-2 strains. Our results provide direct experimental confirmation of the previously inferred gene/enzyme relationship and verify the conclusion that DNA polymerase III is required to replicate the genome. We isolated DNA polymerase III from two cdc2-2 strains, one containing the wild-type allele for DNA polymerase I (CDC17) and the other a mutant DNA polymerase I allele (cdc17-1). Yields from cdc2-2 cells of both DNA polymerase III activity and an associated 3'-5'-exonuclease activity [exonuclease III; Bauer et al. (1988) J. Biol. Chem. 263, 917-924] were decreased relative to yields from CDC2 cells. DNA polymerase III activity from cdc2-2 strains is thermolabile, displaying at least a 4-fold reduction in half-life at 44 degrees C. The activity is also labile at 37 degrees C, a temperature which is restrictive for growth of cdc2-2 but not CDC2 strains. At 23 degrees C, a temperature which is permissive for growth of both cdc2-2 and CDC2 strains, the mutant and wild-type DNA polymerase III activities display equal stability. These observations provide a demonstrable biochemical basis for the thermosensitive phenotype of cdc2-2 cells.

Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.

We have remodeled the gene coding for beta-lactamase by replacing DNA at the active site with random nucleotide sequences. The oligonucleotide replacement (Phe66XXXSer70XXLys73) preserves the codon for the active serine-70 but also contains 15 base pairs of chemically synthesized random sequences that code for 2.5 x 10(6) amino acid substitutions. From a population of Escherichia coli infected with plasmids containing these random inserts, we have selected seven new active-site mutants that render E. coli resistant to carbenicillin and a series of related analogues. Each of the new mutants contains multiple nucleotide substitutions that code for different amino acids surrounding serine-70. Each of the mutants exhibits a temperature-sensitive beta-lactamase activity. This technique offers the possibility of constructing alternative active sites in enzymes on the basis of biological selection for functional variants.

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.