Molecular devices for high fidelity of DNA replication and gene expression

Abstract

Certain types of DNA lesions, produced through cellular metabolic processes and also by external environmental stresses, are responsible for the induction of mutations as well as of cancer. Most of these lesions can be eliminated by DNA repair enzymes, and cells carrying the remaining DNA lesions are subjected to apoptosis. The persistence of damaged bases in RNA can cause errors in gene expression, and the cells appear to possess a mechanism which can prevent damaged RNA molecules from entering the translation process. We have investigated these processes for high fidelity of DNA replication and gene expression, by using both biochemical and genetic means. We herein describe (1) the molecular mechanisms for accurate DNA synthesis, (2) mammalian proteins for sanitizing the DNA precursor pool, (3) error avoidance mechanisms for gene expression under oxidative stress, and (4) the roles of DNA repair and apoptosis in the prevention of cancer.

Keywords: DNA repair; DNA replication; apoptosis; carcinogenesis; gene expression; mutagenesis.

Fig. 1.

Control of spontaneous mutagenesis in E. coli. Degrees of contribution of each step were estimated from mutation frequencies of mutants defective in particular gene functions. Note that some of mutations in a single allele sometimes affect more than one step. These values should be taken as rough estimates, in order to grasp the whole scheme.

Fig. 2.

Actions of mutT, mutM and mutY mutators. A. model for 8-oxoguanine-related mutagenesis. G₀ denotes 8-oxoguanine. B. Types of base substitutions. Base substitution mutations can be classified into transitions, as indicated by black arrows in the center region, and transversions, shown by four arrows in the rectangular form, according to orientations of the base changes. Among the four types of transversions (AT→CG, AT→TA, GC→TA, GC→CG) and two types of transitions (AT→GC, CG→TA), mutT mutant induces only AT→CG transversion while mutM and mutY yield specifically GC→TA transversion. C. The types of mutations arisen in mutants defective in one or more of the mutT, mutM and mutY genes.

Fig. 3.

Comparison of the amino acid sequences of MutT-related proteins. The 23-residues of the MutT signatures are shown below. The residues conserved are indicated in bold letters, and the essential residues for MutT catalytic activity are indicated by dots above the columns.

Fig. 4.

The formation and elimination of 8-oxoguanine-containing deoxyribonucleotides in mammalian cells. The interconversion of oxidized guanine nucleotides, produced by oxidative stress (O^.), is shown in the shaded area. (1) DNA polymerase, (2) nucleoside triphosphatase, (3) nucleoside diphosphate kinase, (4) guanylate kinase.

Fig. 5.

Partial phenotypic suppression of an amber mutation of the lacZ gene of E. coli by expression of cDNA for human MutT-related proteins. A. model for phenotypic suppression. E. coli cells carrying amber mutation in the lacZ gene cannot produce an active β-galactosidase (b). Under the mutT⁻ background, these cells would accumulate 8-oxoGTP (G₀TP), allowing 8-oxoguanine misincorporation into messenger RNA. This would suppress the amber mutation, thereby producing a small but significant amount of β-galactosidase protein. B. β-galactosidase activity of mutT⁺ and mutT ⁻ cells with or without human cDNA. The data were taken from ref. .

Fig. 6.

A model for preventing erroneous protein synthesis by exclusion of 8-oxoguanine-containing ribonucleotides and RNA.

Fig. 7.

The survivals of the mice given various doses of MNU. Six-week-old mice were given various doses of MNU i. p. For each dose 7 to 12 mice were used and data on survivors at 30 days after the administration were plotted.

Fig. 8.

Effects of MNU on the induction of mutation and tumor under various genetic backgrounds. Dotted bars represent non-treated control values, while hatched bars indicate values after MNU treatment (1 mM MNU for mutant frequency assay and 30 mg of MNU/kg of body weight for tumor formation). Data were from ref. , , .

Fig. 9.

Roles of DNA repair and apoptosis in preventing mutations and cancer.