Base Mispairs Research Articles

Site specificity of incisions at G:T and O6-methylguanine:T base mismatches in DNA by human cell-free extracts.

Cell-free extract from human tumor cell line A1235 (lacking O6-methylguanine-DNA methyltransferase) was employed to compare incision at G:T base mispairs with that at O6-methylguanine (m6G):T pairs at two different sites (sites 20 and 25) in 45-bp heteroduplexes. To study the effect of neighboring bases on the activity(ies), the base pair immediately 5' to the mismatched G at each site was varied to provide four contexts: CpG:T, TpG:T, ApG:T, and GpG:T (and two analogous series for m6G:T pairs). At site 20, cell-free extract produced observable incision only in the 45-bp DNA with the G:T mispair in the CpG:T context, giving a product with incisions immediately 5' and 3' to the mismatched T. We observed incision of neither the strand containing the mismatched G nor the DNAs with the site 20 ApG:T, GpG:T, and TpG:T mismatches. By contrast, at site 25, incision specificity was different. All four G:T mismatched DNAs were incised, and the ApG:T-25, GpG:T-25, and TpG:T-25 DNAs were incised 1-3 bonds 3' to the mismatched T, while similar in other respects to the CpG:T-25 DNA, which showed a pattern like the CpG:T-20 DNA. CpG:T-20 specific incision activity in the extract was strongly inhibited by both CpG:T (sites 20 and 25) DNAs, but at least 10-fold more poorly by DNAs with Apg:T-25 and GpG:T-25 pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoguanosine Substitution of Conserved Adenosines in the Hammerhead Ribozyme

Isoguanosine has been incorporated into a 34-mer hammerhead ribozyme by the solid-phase phosphoramidite method, using an acetamidine base protecting group. The activity of the hammerhead ribozyme when singly mutated to isoguanosine at the adenosine positions 6, 9, and 13 was 1-2-fold less than the wild-type activity. Mutations to 2-aminopurine ribonucleoside at positions 9 and 13 were 5-fold reduced in activity, but that at position 6 was approximately 30-fold reduced. These results support the view that the 6-amino functions of A6, A9, and A13 are not very important for catalysis. The 2-position of A6 tolerates a carbonyl function but not an amino group, whereas A9 and A13 tolerate both functional groups. The tolerance of a 2-amino group at A9 and A13 makes G(anti)/A(anti) Watson-Crick type base mispairing for G12/A9 and A13/G8 unlikely.

Defective transformation of chromosomal markers in DNA polymyerase I mutants of the radioresistant bacterium Deinococcus radiodurans

The transformation efficiency of six independently slected chromosomal markers (four for rifampicin resistance and two for acriflavine resistance) was found to be reduced by about 3 logs in a Deinococcus radiodurans strain that was isogenic with wild type except for an insertional mutation in the pol gene that eliminated DNA polymerase I activity (strain 6R1A). D. radiodurans strains UV17 and 303, previously by chemical mutagenesis, were determined to be partially deficient in DNA Pol I activity as assessed in a permeabilized cell system. Both UV17 and 303 demonstrated intermediate transforming efficiencies that correlated with their levels of residual polymerase activity. The transformation efficiency of strain 6R1A could be greatly restored by expression of clones E. coli DNA Pol I, but not to wild-type levels. Plasmid transfer and chromosomal duplication insertion were not substantially affected by lack of DNA Pol I activity. D. radiodurans is known to possess extraordinarily efficient repair pathways for DNA damage, and is refractory to DNA damage-induced mutagenesis caused by numerous agents, including several that cause base mispairing. We suggest that D. radiodurans may differ from other naturally transformable bacteria in that DNA Pol I is needed to efficiency convert most drug-resistance markers. This unusual mechanism may be reuqired to acoomplish chromosomal conversion prior to correction of donor DNA by this organism's efficient repair pathways.

Mammalian topoisomerase I has base mismatch nicking activity.

The all-type nicking enzyme (ATE) from human HeLa cells or calf thymus can nick DNA at the first phosphodiester bond 5' to all 8 possible mismatched bases. The strand disparity of this nicking is influenced by the neighboring nucleotide sequences. After nicking, the ATE covalently binds to the 3' end of the DNA product to form a cleavable complex, whose formation is insensitive to camptothecin, a specific inhibitor of eukaryotic topoisomerase I (Topo-I). During the purification of ATE from calf thymus, a Mg(2+)-independent relaxation activity, characteristic of eukaryotic Topo-I, copurifies with the mismatch-nicking activity. The ATE from calf thymus may be a breakdown product of Topo-I. N-terminal amino acid analysis indicates that one of the polypeptides with ATE activity contains the C-terminal portion of Topo-I. Moreover, active human Topo-I, expressed as a fusion protein in Escherichia coli, is also capable of nicking all 8 base mispairs in the absence of Mg2+. This mismatch-specific nicking activity may be a novel property of the mammalian Topo-I.

Origins of spontaneous base substitutions

Although simple to understand, satisfying to the imagination, and compelling as a teaching aid for beginning students, the evidence is mounting that the tautomeric shift is neither a common nor a likely origin for spontaneous base substitutions. Indeed, other sources, such as ionized base mispairings with nonionized bases, wobble of a bases in the DNA, and transient misalignment of bases causing dislocations at pairing sites, have been shown to induce spontaneous base substitutions. On the other hand, among the 4 common bases in DNA, no experimental evidence exists that tautomeric shifts can induce mutations.

G-->A hypermutation of the human immunodeficiency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription.

The quasispecies model for RNA viruses predicts the existence of a replication error threshold beyond which there is a melting or total loss of sequence information. Retroviral G-->A hypermutation is probably an example. Here it is shown that G-->A transitions may occur in both GpG and GpA dinucleotide contexts. Transitions in GpG preferentially occur via base mispairing at the ends of runs of G residues, whereas G-->A transitions within GpA may result from temporary dislocation of the primer and template strands by a single base. The two circumstances may be related by the local dCTP substrate concentration. An in vitro elongation assay shows that primer/template dislocation is more frequent for the human immunodeficiency virus type 1 reverse transcriptase than for murine or avian retroviral enzymes. Taken together these data suggest that G-->A hypermutation is an example of induced mutation whereby the viral reverse transcriptase is forced into making errors by imbalances in the intracellular dCTP concentration.

Oxidative DNA damage — The effects of certain genotoxic and operationally non-genotoxic carcinogens

A wide variety of oxidative DNA lesions are commonly present in untreated human and animal DNA. One of these lesions, 8-hydroxydeoxyguanosine, has been shown to lead to base mispairing (mutation) on DNA replication. Other lesions remain to be investigated in this respect. Oxidative DNA lesions on cell replication may, in appropriate circumstances, lead to proto-oncogene activation. Oxidative DNA damage, on fixation, may also lead to cytotoxicity followed by regenerative proliferation. The probable or possible importance of oxidative DNA damage is reviewed for various classes of carcinogens and natural processes, including metal ions, high-energy radiation, miscellaneous chemicals, tumor-promoting agents, polyhydroxyphenols/quinones, lipid metabolism, peroxisome proliferators and thyroid function. It is concluded that although the evidence needs considerable strengthening in many of these examples, the available information indicates the potential importance of oxidative DNA damage in the induction of tumors by these agents. It is also possible that non-cancerous degenerative diseases associated with aging are the result of the accumulation of lesions resulting from unrepaired oxidative DNA damage.

Studies of the structure and stability of base pair mismatches, base pairs involving modified bases, and DNA-drug complexes

Of the DNA bases, the hydrogen bonding characteristics of guanine (G) are complementary only to those of cytidine (C) and, similarly, the hydrogen bonding characteristics of adenine (A) are complementary only to those of thymine (T) (Fig. 1). This is the fundamental basis of Watson-Crick base pairing in DNA and ensures the extremely high fidelity of DNA replication which is essential in maintaining the integrity of living organisms. However, point mutations can, and do, occur and are the result of the formation of base pair mismatches between the native DNA bases during DNA synthesis or the misinsertion of a base by DNA polymerases caused by the presence of modified DNA bases in the template strand 9 There are two types of point mutation, known as transitions and transversions (Fig. 2). Transition mutations arise as a result of purine, pyrimidine mispairs and in this case one purine is replaced by the other purine or one pyrimidine by the other pyrimidine. In transversion mutations a purine is substituted by a pyrimidine and vice versa 9 This type of mutation is a consequence of purine.purine or pyrimindine.pyrimidine mismatches. In order for a base mispair to survive until the next round of DNA replication it must evade two checking procedures. The first occurs as each nucleoside triphosphate is incorporated into the newly synthesized DNA while the second, post-synthetic, step is a proofreading

Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine

To test whether ionized base pairs influence polymerase-catalyzed misinsertion rates, we measured the efficiency of forming 5-bromouracil (B), 5-fluorouracil (F), and thymine base pairs with guanine and adenine as a function of pH using avian myeloblastosis reverse transcriptase. When B, F, and T were present as dNTP substrates, misincorporation efficiencies opposite G, normalized to incorporation of C opposite G, increased by about 20-, 13-, and 7-fold, respectively, as reaction pH increased from 7.0 to 9.5. Incorporation efficiencies to form the correct base pairs, B.A and F.A, normalized to T.A, decreased by 4- and 8-fold, respectively, with increasing pH. The effects of pH on misincorporation efficiencies were about 10-fold greater when B, F, and T were present as template bases. The relative misincorporation efficiencies of G opposite template B, F, and T, normalized to incorporation of A opposite B, F, and T, increased by about 430-, 370-, and 70-fold, respectively, as pH was increased from 6.5 to 9.5, while correct incorporation of A opposite template B and F decreased about 10-fold over the same pH range. Plots depicting incorrect and correct incorporation efficiencies versus pH were fit to a pH titration equation giving the fraction of ionized base as a function of pH. We conclude that avian myeloblastosis reverse transcriptase forms B.G and F.G mispairs in an ionized Watson-Crick conformation in preference to a neutral wobble structure containing favored keto tautomers of B or F. Although participation of disfavored enol tautomers in enzyme-catalyzed base mispair formation cannot be ruled out, the results are inconsistent with the "standard" disfavored tautomer model of mutagenesis. Instead, the data support a model in which ionization of halouracil bases is primarily responsible for B- and F-induced mutagenesis.

Keto-iminol tautomerism of protonated cytidine monophosphate characterized by ultraviolet resonance Raman spectroscopy: implications of C+ iminol tautomer for base mispairing

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKeto-iminol tautomerism of protonated cytidine monophosphate characterized by ultraviolet resonance Raman spectroscopy: implications of C+ iminol tautomer for base mispairingRoberto Purrello, Marina Molina, Yang Wang, Giulietta Smulevich, Jacques R. Fresco, Thomas G. Spiro, and John FossellaCite this: J. Am. Chem. Soc. 1993, 115, 2, 760–767Publication Date (Print):January 1, 1993Publication History Published online1 May 2002Published inissue 1 January 1993https://doi.org/10.1021/ja00055a056RIGHTS & PERMISSIONSArticle Views387Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (891 KB) Get e-Alerts Get e-Alerts

Computational investigation of the role of hydration in nucleic acid structure and function

AbstractThe results of the Monte Carlo Metropolis simulation of water structure and of the hydration of nucleic acid fragments, complementary base pairs and mispairs, base pair stacks, and duplex fragments have been summarized. Systematic investigations suggest some general conclusions: (1) the hydration shell structure of the major and minor grooves of the duplex depends significantly on DNA conformation (or stack configuration) and nucleotide sequence, while global hydration characteristics (average energy, the number of water–water and water–base H‐bonds) are only slightly dependent on these factors, (2) hydration economy takes place in the B–A transition due to an increase of the number of water molecules forming hydrogen bonds with two or more atoms of bases (water bridging), and (3) the hydration of the duplex could contribute to nucleic acid functioning via water‐bridged mispair formation and stabilization of specific conformations.

Structural basis of DNA mutagenesis

The scope of this review covers those NMR and X-ray crystallographic structural analyses that have been carried out in recent months on a number of mutagenic lesions, mismatched base pairs and a mutational hotspot in synthetic DNA duplexes. The structure of DNA duplexes containing the mutagenic bases O(6)-methyl deoxyguanosine, O(6)-ethyl deoxyguanosine, 8-hydroxydeoxyguanosine and 1,N(6)-ethenodeoxyadenosine are discussed. Recent studies on DNA duplexes containing G·A mismatched base pairs are also reviewed. Finally, an important study of a dodecamer containing a sequence corresponding to a hotspot for frameshift mutagenesis is described. This structure contains a novel type of groove backbone interaction and novel base mispairing which suggest a molecular model for frameshift substitution mutagenesis.

Ionizing radiation and genetic risks I. Epidemiological, population genetic, biochemical and molecular aspects of mendelian diseases

This paper reviews the currently available information on naturally occurring Mendelian diseases in man; it is aimed at providing a background and framework for discussion of experimental data on radiation-induced mutations (papers II and III) and for the estimation of the risk of Mendelian disease in human populations exposed to ionizing radiation (paper IV). Current consensus estimates indicate that a total of about 125 per 10 4 livebirths are directly affected by one or another naturally occurring Mendelian disease (autosomal dominants, 95/10 4; X-linked ones, 5/10 4; and autosomal recessives, 25/10 4). These estimates are conservative and take into account conditions which are very rare and for which prevalence estimates are unavailable. Most, although not all, of the recognized “common” dominants have onset in adult ages while most sex-linked and autosomal recessives have onset at birth or in childhood. Autosomal dominant and X-linked diseases (i.e., the responsible mutant alleles) presumed to be maintained in the population due to a balance between mutation and selection are the ones which may be expected to increase in frequency as a result of radiation exposures. Viewed from this standpoint, the above assumption seems safe only for a small proportion of such diseases; for the remainder, there is no easy way to discriminate between different mechanisms that may be responsible or to rigorously exclude some in favor of some others. Mutations in genes that code for enzymic proteins are more often recessive in contrast to those that code for non-enzymic proteins, which are more often dominant. At the molecular level, with recessives, a wide variety of changes is possible and these include specific types of point mutations, small and large intragenic deletions, multilocus deletions and rearrangements. In the case of dominants, however, the kinds of recoverable point mutations and deletion-type changes are less extensive because of functional constraints. The mutational potential of genes varies, depending on the gene, its size, sequence content and arrangement, location and its normal functions, and can be grouped into three groups: those in which only point mutations have been found to occur, those in which only deletions or other gross changes have been recovered and those in which both kinds of changes are known. Molecular data are available for about 75 Mendelian conditions and these suggest that in approximately 50% of them, the changes categorized to date are point mutations and in the remainder, intragenic deletions or other gross changes; there does not seem to be any fundamental difference between dominants and recessives with respect to the underlying molecular defect. Onset age for Mendelian diseases appears related to the normal function disrupted or abolished by the mutation and not necessarily to the underlying molecular change. Point mutations do not appear to be distributed at random throughout the gene; CpG dinucleotide sequences, when present in the gene, provide “hot-spots” for transition-type mutations, but not all transitions occur at CpG sequences. The breakpoints involved in intragenic deletions also are not distributed at random within the gene. In cases analyzed, the length of the deletion per se is not correlated with the severity of the clinical effect. The data from a number of well-analyzed gene deletions are consistent with mechanisms that assume base mispairing between repeat sequences and slippage during replication, homologous unequal recombination between evolutionarily related genes, homologous unequal recombination between repetitive sequences such as Alu and non-homologous recombination. Not all deletions involve Alu sequences. There is circumstantial evidence supporting the hypothesis that repetitive sequences may play an important role in chromosome pairing; if true, the deletions and duplications that have been found to be associated with some diseases may represent the inevitable by-products of occasional mispairing.

Time-dependent changes in gene conversion inAscobolus immersus

SummaryThe stability of conversion frequencies with time was investigated inAscobolus immersus. There were usually marked reductions in gene conversion frequencies at locuswlas crosses matured, to about one third of the initial values. This applied to all sixwlalleles tested, irrespective of their molecular nature, and at all temperatures used. Asci dehiscing early from an apothecium had much higher conversion frequencies than those dehiscing late, but there were no differences between apothecia maturing at different times within a cross. Alleles at four loci unlinked towlwere also tested. All four showed significant changes, though not in all crosses; three loci showed decreases in conversion frequency with time, while one showed an increase. The relative frequencies of different conversion classes often changed with time. These changes appear to result from alterations in locus-specific recombination initiation and in repair of base mispairs in hybrid DNA, not from differential maturation rates of different ascal segregation classes. These effects could cause misinterpretation of quantitative tests of recombination models from gene conversion data.

Frequent activation of the Ki‐ras oncogene at codon 12 in N‐methyl‐N‐nitrosourea‐induced rat prostate adenocarcinomas and neurogenic sarcomas

Rat neoplasms induced by methylating carcinogens frequently contain ras genes activated by a single point mutation. Rat prostatic tumors induced by a combination of a single injection of N-methyl-N-nitrosourea (MNU) and long-term treatment with testosterone were examined for the presence of such activating point mutations in ras genes. These tumors, which arose exclusively in the dorsolateral prostate, included both adenocarcinomas and sarcomas. Activating mutations in codon 12 of the Ki-ras gene were found in 7 of 10 carcinomas and 4 of 5 sarcomas, using selective oligonucleotide hybridization analysis of DNA amplified by the polymerase chain reaction (PCR). However, no mutated Ha-ras oncogenes were detected. The presence of PCR-engineered Hphl restriction sites created by the existence of a G35----A mutation in the rat Ki-ras oncogene identified the mutation as a GC----AT transition at the second position of codon 12. Production of O6-methylguanine adducts in the Ki-ras codon 12 followed by base mispairing during replicative DNA synthesis is thus the likely molecular mechanism of initiation of prostatic carcinogenesis by MNU in the rat. Three of the four sarcomas positive for the Ki-ras G35----A mutation were immunohistochemically defined as of Schwann cell origin, indicating that involvement of the ras gene family is possible in tumorigenesis of this cell lineage. Loss of the wild-type Ki-ras allele was also observed in all four of these sarcomas.

Role of Clara Cells and Type II Cells in the Development of Pulmonary Tumors in Rats and Mice Following Exposure to a Tobacco-Specific Nitrosamine

The role of the Clara and type II cell in the development of pulmonary tumors in the A/J mouse and Fischer rat was investigated by determining the relationship of DNA methylation and repair in pulmonary cells to oncogene activation and by characterizing the morphology of pulmonary tumors induced by treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Marked differences in the formation of the promutagenic adduct O6-methylguanine (O6MG) were observed in pulmonary cells following treatment of rats with NNK. Concentrations of this adduct in Clara cells greatly exceeded (3- to 30-fold) those detected in type II cells and whole lung with doses of NNK ranging from 0.1 to 50 mg/kg. In addition, very low rates of repair of this adduct were detected in Clara cells, whereas efficient adduct removal occurred in type II cells. The importance of this adduct and the role of cell specificity was suggested by the fact that a strong correlation was observed between the concentration of O6MG in Clara cells and tumor incidence in the Fischer rat with doses of NNK ranging from 0.03-50 mg/kg. In contrast, no differences in adduct concentration between type II and Clara cells from A/J mice were observed under conditions resulting in pulmonary tumor formation. Activation of the K-ras gene was detected in lung tumors from A/J mice. This gene was activated by a mutation in codon 12 involving a GC to AT transition (GGT to GAT) and is consistent with base mispairing produced by the formation of O6MG. Activation of this gene was not associated with lung tumor formation in the Fischer rat. DNA from rat lung tumors did induce tumors in the nude mouse carcinogenicity assay. In addition, rat repetitive sequences were detected in DNA isolated from these nude mouse tumors. In spite of the cell selectivity for DNA methylation in Clara cells from rat and the relationship between O6MG formation and tumorigenicity, early proliferative lesions observed in both mice and rats involved the alveolar areas. Ultrastructural examination of these lesions and adenomas revealed morphologic features characteristic of the type II cell. Thus the lack of agreement between biochemical and morphological findings makes it difficult to hypothesize a cell of origin for the pulmonary neoplasms induced by NNK. However, these studies indicate that the concentration of O6MG in Clara cells is an excellent indicator of the carcinogenic potency of NNK in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of DNA methylation in the activation of proto-oncogenes and the induction of pulmonary neoplasia by nitrosamines

The relationships between DNA methylation and repair induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to the activation of proto-oncogenes and the induction of pulmonary neoplasia by this carcinogen is described. The formation of the O 6-methylguanine (O 6MG) adduct following metabolic activation of NNK appears to be a major factor in the induction of lung tumors in both rats and mice and in the activation of the K- ras oncogene in lung tumors from A/J mouse. The potent carcinogenicity of NNK in the rat lung correlated strongly with cell specificity for formation and persistence of the O 6MG adduct in the Clara cells. This conclusion was supported by studies with nitrosodimethylamine (NDMA), a weak carcinogen in the rodent lung. Treatment with NDMA was not associated with any pulmonary cell specificity for DNA methylation. The high affinity for activation of NNK compared to NDMA was ascribed to a difference in cytochrome P-450 isozymes involved in the activation of these two nitrosamines. In the A/J mouse, the induction of pulmonary tumorigenesis involved direct genotoxic activation of the K- ras proto-oncogene as a result of the base mispairing produced by formation of the O 6MG adduct. In contrast, the induction of pulmonary tumors in the rat by NNK does not appear to involve the ras pathway. It is apparent that different molecular mechanisms are involved in the development of pulmonary tumors by NNK in the mouse and rat. The studies described in this paper illustrate the utility of performing dose-response experiments and the quantitation of DNA methylation and repair in not only target tissues but also target cell types. The fundamental knowledge gained from unraveling the mechanism of carcinogenesis by NNK could lead ultimately to the identification of factors important in the development of human lung cancer.

Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase.

A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.

Repair of base–base mismatches in simian and human cells

Mismatched heteroduplexes arise as intermediates of several dissimilar genetic processes. The outcome of these genetic events will therefore be influenced by the efficiency and specificity of mismatch repair. We have studied the correction of base-base mispairs in simian and human fibroblasts by transfecting the cells with derivatives of SV40 DNA, each harboring a single mispair in a defined orientation. Analysis of plaques revealed that correction efficiencies for homomispairs followed the pattern G.G greater than C.C greater than or equal to A.A greater than T.T. Repair bias was influenced by flanking sequences. Correction efficiences for heteromispairs followed the pattern of G.T greater than A.C greater than C.T greater than A.G and repair favored the retention of G + C by a substantial margin. This repair specificity could lead to a gene conversion bias favoring the accumulation of G + C in sequences subject to high levels of recombination or unequal exchange.

Effect of pH on the base-mispairing properties of 5-bromouracil during DNA synthesis.

We have utilized an electrophoretic assay of misincorporation to investigate the possibility that ionization of 5-bromouracil (BU) may play a role in its mispairing during DNA synthesis in vitro. We examined the effects of increasing pH on the relative rates of formation of BU.G and T.G mispairs during chain elongation catalyzed by various DNA polymerases. For the Klenow fragment of Escherichia coli DNA polymerase I, increasing pH facilitated BU.G mispair formation (relative to T.G mispairing) when BU was present in the template strand. This effect showed a strong dependence on sequence context. Increasing pH had little effect on the relative rate of misincorporation of BrdUMP versus dTMP (at template G) by the Klenow polymerase. Misincorporation opposite template BU residues catalyzed by Maloney murine leukemia virus DNA polymerase and DNA polymerase beta (Novikoff hepatoma) also increased with pH, but for these two enzymes, there was no apparent dependence on sequence context. With T4 DNA polymerase and E. coli DNA polymerase III holoenzyme, a similar occurrence of BU.G and T.G mispairing during polymerization was observed, whether BU was present in the template or in the incoming nucleotide, and there was little effect of pH. The results reported here are consistent with a mispairing mechanism for template BU wherein the anionic form of the base mispairs with G.