DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.     

Single-strand conformation polymorphism for detection of mutations and base substitutions in phenylketonuria.

In the past few years, more than 20 different mutations have been reported in hyperphenylalaninemias. In southwestern Europe and Mediterranean countries, however, the mutant genotypes reported account for only a fraction (27%) of all mutant alleles at the phenylalanine hydroxylase (PAH) locus, and most of the mutations causing the disease remain unknown. In order to develop a strategy for rapid detection of mutation-containing exons, we applied the single-strand conformation-polymorphism (SSCP) technique to exons 3, 5, 7, and 12 of the PAH gene. We observed five abnormal patterns of migration in mutant PAH genes, and we consistently found base substitutions in the corresponding exons, with no false-positive results. By this procedure, two novel putative mutations were detected in the seventh exon of the PAH gene, (A259V and Y277D) and we were able to demonstrate that the delta I94, R158Q, R408W, and E280K mutations were easily detectable by the SSCP technique. This procedure is therefore of particular interest for rapid detection of mutation-containing exons and for determination of further genotype-phenotype correlations in hyperphenylalaninemias.     

DNA base modifications in chromatin of human cancerous tissues.

Free radical-induced damage to DNA in vivo is implicated to play a role in carcinogenesis. Evidence exists that DNA damage by endogenous free radicals occurs in vivo, and there is a steady-state level of free radical-modified bases in cellular DNA. We have investigated endogenous levels of typical free radical-induced DNA base modifications in chromatin of various human cancerous tissues and their cancer-free surrounding tissues. Five different types of surgically removed tissues were used, namely colon, stomach, ovary, brain and lung tissues. In chromatin samples isolated from these tissues, five pyrimidine-derived and six purine-derived modified DNA bases were identified and quantitated by gas chromatography/mass spectrometry with selected-ion monitoring. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, 5-hydroxycytosine, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, xanthine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. These compounds are known to be formed typically by hydroxyl radical attack on DNA bases. In all cases, elevated amounts over control levels of modified DNA bases were found in cancerous tissues. The amounts of modified bases depended on the tissue type. Lung tissues removed from smokers had the highest increases of modified bases above the control levels, and the highest overall amounts. Colon cancer tissue samples had the lowest increases of modified bases over the control levels. The results clearly indicate higher steady-state levels of modified DNA bases in cancerous tissues than in their cancer-free surrounding tissues. Some of these lesions are known to be promutagenic, although others have not been investigated for their mutagenicity. Identified DNA lesions may play a causative role in carcinogenesis.     

Randomness of base substitution mutations induced in the lacI gene of Escherichia coli by ionizing radiation

The lacI system of Escherichia coli provides a method for monitoring mutational events at a large number of sites. Using this system, we have previously determined the mutational spectra for gamma-ray and beta-particle emissions resulting from the decay of tritium. Analysis of these mutational spectra reveals that base substitution mutations induced by ionizing radiation are distributed nearly randomly throughout the lacI gene and include all detectable substitution events. The distribution of ionizing radiation-induced mutagenesis is similar to the low frequency of occurrence mutational events induced by other SOS-dependent mutagens. The lack of an apparent nonrandom or high frequency of occurrence component seen with other SOS-dependent mutagens can be best explained as the result of the random interaction of ionizing radiation with the DNA bases leading to production of a variety of base substitutions.     

Induction of base substitution mutations by aflatoxin B1 is mucAB dependent in Escherichia coli.

Recovery of aflatoxin B1-induced base substitution mutations in Escherichia coli was almost completely dependent on the presence of the SOS-mutagenesis-enhancing operon mucAB+; the normal E. coli analog, umuDC+, was not sufficient. Yet aflatoxin B1 induced the SOS response, including the umuDC operon, as well as did UV light. Neither preinduction of the SOS response nor the presence of additional copies of umuDC+ allowed the recovery of aflatoxin B1-induced base substitutions. Thus, the premutagenic DNA lesions induced by aflatoxin B1 reveal a functional difference between UmuDC and MucAB. We estimate that in the presence of MucAB the probability that aflatoxin B1-induced DNA lesions will be converted into mutations is increased at least 10-fold.     

Deleterious mitochondrial DNA mutations accumulate in aging human tissues.

This paper reviews the current state of knowledge of the contribution of mitochondrial DNA (mtDNA) mutations to the phenotype of aging. Its major focus is on the discovery of deletions of mtDNA which previously were thought to occur only in individuals with neuromuscular disease. One particular deletion (mtDNA4977) accumulates with age primarily in non-dividing cells such as muscle and brain of normal individuals. The level of the deletion rises with age by more than 1000 fold in heart and brain and to a lesser extent in other tissues. In the brain, different regions have substantially different levels of the deletion. High levels of accumulation of the deletion in tissues are correlated with high oxygen consumption. We speculate that oxidative damage to mtDNA may be 'catastrophic'; mutations affecting mitochondrially encoded polypeptides involved in electron transport could increase free radical generation leading to more mtDNA damage.     

UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells.

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5'-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.     

Calcitonin receptor-like receptor: identification and distribution in human peripheral tissues

We report here on the characterization and immunohistochemical localization in human tissues of calcitonin receptor-like receptor (CRLR) which was recently found to mediate the effects of both calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM). Western blot analysis using antibodies raised against the first extracellular loop and the carboxy-terminal part of hCRLR, respectively, detected two major bands corresponding to about 70 and 60 kDa in membrane preparations of cultured endothelial cells and numerous organs including lung, heart ventricle and kidney. Immunohistochemical analysis of the cardiovascular system revealed CRLR-like immunoreactivity (CRLR-LI) in the endothelium of all blood vessels including large and small arteries, veins and capillaries, and in heart muscle cells and endocardium. The lung showed intense staining over the alveolar capillaries. Within the digestive tract, staining was observed over the cells lining the excretory ducts of the parotid gland, over the epithelium of the fundic glands of stomach, endocrine cells of the duodenum and ileum and some myenteric ganglia. The kidney presented staining of the juxtaglomerular arteries, the glomerular capillaries and chief cells of the collecting duct. Within the endocrine organs, a strong CRLR-LI signal was observed over the Langerhans islets, and weak immunoreactivity in the Leydig cells of testis. Spleen showed intense staining in trabecular veins and sinuses. Macrophages displayed a variable immunoreactivity. Our data demonstrate a wide distribution of CRLR throughout the human body and suggest CRLR to be involved in the mediation of a variety of actions in addition to vascular control.     

Immunohistochemical detection of glycogen phosphorylase isoenzymes in rat and human tissues

Summary Glycogen phosphorylase has at least three isoenzymes, i.e. muscle-, liver-, and brain-types. Antibodies have been raised against highly purified isoenzymes from rat muscle, liver and brain and found to react specifically to extracts from human muscle, liver and brain, respectively. Using these antibodies and the unlabelled antibody—enzyme method, each of the three isoenzymes has been localized in both rat and human tissues.     

Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ～70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ～30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ～40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.     

Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome

Mutations in the rpoB gene of Escherichia coli result in resistance to the antibiotic rifampicin (Rif(r)) by altering the beta subunit of RNA polymerase. Previous studies have identified 39 single base substitutions in the rpoB gene that lead to Rif(r) at 37 degrees C and an additional two mutations that result in temperature sensitive cells. We have extended this work and identified an additional 30 single base substitutions that result in the Rif(r) phenotype. With these mutations the rpoB/Rif(r) system now allows the monitoring of 69 base substitutions at 37 degrees at 37 sites (base pairs) distributed among 24 coding positions. Each of the six possible base substitutions is represented by 8-17 mutations. More than 90% of the mutations are within a small enough region of the rpoB gene to allow PCR amplification with a single pair of oligonucleotide primers, followed by sequencing with a single primer, leading to rapid analysis of numerous mutations. The remaining mutations can be monitored using an additional primer pair. To calibrate this system we sequenced over 500 mutations in rpoB occurring spontaneously or generated by different mutagens and mutators with known specificity. These results show that rpoB/Rif(r) is an accurate and easy to employ detection system, and offers the advantage of allowing analysis of mutations occurring on the chromosome rather than on an extrachromosomal element. The mutS, mutT, mutY, M mutators, as well as the mutagenic agents ethyl methanesulfonate (EMS), ultraviolet (UV) irradiation, 2-aminopurine (2AP), 5-azacytidine (5AZ), and cisplatin (CPT) gave results predicted by their characterized specificities. The number of different sequence contexts is sufficient to reveal significant hotspots among the spontaneous mutS, 2-aminopurine, ultraviolet light, 5-azacytidine, and cisplatin mutational spectra. The cisplatin distribution is particularly striking, with 68% of the mutations resulting from an A:T-->T:A transversion at a single site. Because of the conservation of key regions of RNA polymerase among many microorganisms, using the Rif(r)/rpoB system may be a general method for studying mutational processes in microorganisms without well developed genetic systems.     

Ataxia-telangiectasia: identification and detection of founder-effect mutations in the ATM gene in ethnic populations.

To facilitate the evaluation of ATM heterozygotes for susceptibility to other diseases, such as breast cancer, we have attempted to define the most common mutations and their frequencies in ataxia-telangiectasia (A-T) homozygotes from 10 ethnic populations. Both genomic mutations and their effects on cDNA were characterized. Protein-truncation testing of the entire ATM cDNA detected 92 (66%) truncating mutations in 140 mutant alleles screened. The haplotyping of patients with identical mutations indicates that almost all of these represent common ancestry and that very few spontaneously recurring ATM mutations exist. Assays requiring minimal amounts of genomic DNA were designed to allow rapid screening for common ethnic mutations. These rapid assays detected mutations in 76% of Costa Rican patients (3), 50% of Norwegian patients (1), 25% of Polish patients (4), and 14% of Italian patients (1), as well as in patients of Amish/Mennonite and Irish English backgrounds. Additional mutations were observed in Japanese, Utah Mormon, and African American patients. These assays should facilitate screening for A-T heterozygotes in the populations studied.     

Common polymorphisms and somatic mutations in human base excision repair genes in ovarian and endometrial cancers

The purpose of this study was to determine whether the human APEX and OGG1 genes, encoding proteins important in base excision repair (BER) of DNA, contain nucleotide sequence polymorphisms or are mutated somatically in tumors from women diagnosed with ovarian or endometrial cancer. Based upon the analysis of germline DNA from 83 individuals, 63 with ovarian cancer and 20 with endometrial cancer, we found two missense polymorphisms in APEX (Q51H and D 148E) and two missense (A3P and S326C) and one intronic (Exon 5-15 bp) polymorphism in OGG1. The frequencies of the various alleles (in the ovarian and endometrial cancer patients combined) were 4.8% for 51-His and 56.2% for 148-Glu in APEX, and 1.0% for 3-Pro and 20.0% for 326-Cys in OGG1. Somatic mutations in APEX (P112L, W188X and R237C) were identified in three of 20 endometrial tumors, but no mutations were identified in APEX in 43 ovarian tumors, or in OGG1 at either tumor site. Given the crucial role of the APEX and OGG1 proteins in BER of oxidative DNA damage, the identified polymorphisms are good candidates for genetic epidemiologic studies of cancer susceptibility, while the finding that three of 20 (15%) endometrial tumors have somatic mutations in APEX suggests that inactivation of the BER pathway is important for the development of endometrial cancer in at least a subset of cases.     

Neighboring base effects on substitution rates in pseudogenes

Substitution rates in pseudogenes can be used to estimate the frequencies of different types of mutation on the assumption that pseudogenes are not subject to selective constraints. These rates are used here to investigate the effect of neighboring bases on mutation rates. There is a marked increase in the frequency of transitions, though not of transversions, from the doublet CG. There are also some smaller effects of neighboring bases on the frequencies of transitions from adenine and thymine. The results are used to predict dinucleotide frequencies in a stretch of DNA subject to no selective constraints and to investigate the possibility of non-randomness in the usage of stop codons.      

Immunohistochemical detection of lactoferrin of human milk in the tissues of the human and the fetus

By means of the indirect immunoperoxidase method in deparaffinized slices a comparative investigation on distribution of the female milk lactoferrin (LF) in tissues of the mature person and in the fetus has been performed. LF is revealed in cells of the neutrophil line of the mature person and of the fetus and in the secretory epithelium of some organs of the mature person (in the mammary, submandibular and parotid salivary glands, in the bronchial mucous membrane glands in the fundal and pyloric glands of the stomach). In all the cases investigated LF is revealed in the cells producing serous secrete: in the cells of the serous terminal parts and in the serous semilunar mixed terminal parts of the salivary glands, in the serous cells of the bronchial glands and in the chief cells of the gastric mucous membrane glands. In the fetal secretory epithelium of the organs LF is not found. As LF is revealed in the secretory epithelium of the mature person and is not revealed in the corresponding epithelium of the fetus, it should be considered as a marker of the cells, that reach certain degree of differentiation.     

The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations

The frequency and specificity of mutations produced in vitro by eucaryotic DNA polymerase-beta have been determined in a forward mutation assay using a 250-base target sequence in M13mp2 DNA. Homogeneous DNA polymerase-beta, isolated from four different sources, produces mutations at a frequency of 4-6%/single round of gap-filling DNA synthesis. DNA sequence analyses of 460 independent mutants resulting from this error-prone DNA synthesis demonstrate a wide variety of mutational events. Frameshift and base substitutions are made at approximately equal frequency and together comprise about 90% of all mutations. Two mutational "hot spots" for frameshift and base substitution mutations were observed. The characteristics of the mutations at these sites suggest that certain base substitution errors result from dislocation of template bases rather than from direct mispair formation by DNA polymerase-beta. When considering the entire target sequence, single-base frameshift mutations occur primarily in runs of identical bases, usually pyrimidines. The loss of a single base occurs 20-80 times more frequently than single-base additions and much more frequently than the loss of two or more bases. Base substitutions occur at many sites throughout the target, representing a wide spectrum of mispair formations. Averaged over a large number of phenotypically detectable sites, the base substitution error frequency is greater than one mistake for every 5000 bases polymerized. Large deletion mutations are also observed, at a frequency more than 10-fold over background, indicating that purified DNA polymerases alone are capable of producing such deletions. These data are discussed in relation to the physical and kinetic properties of the purified enzymes and with respect to the proposed role for this DNA polymerase in vivo.     

Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38–82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.