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At least 217 records · Page 12Linked to original sources
PubMed2002

Peroxynitrite-induced reactions of synthetic oligo 2'-deoxynucleotides and DNA containing guanine: formation and stability of a 5-guanidino-4-nitroimidazole lesion.

Peroxynitrite is a strong oxidizing agent that is formed in the reaction of nitric oxide and superoxide anion. It is capable of oxidizing and nitrating a variety of biological targets including DNA, and these modifications may be responsible for a number of pathological conditions and diseases. A recent study showed that peroxynitrite reacts with 2',3',5'-tri-O-acetylguanosine to yield a novel compound, tri-O-acetyl-1-(beta-D-erythro-pentafuranosyl)-5-guanidino-4-nitroimidazole, and, unlike other peroxynitrite-mediated guanine oxidation products, it is a stable and significant component formed even at low peroxynitrite concentrations. In this work, we studied the in vitro formation of the guanine-derived product, 5-guanidino-4-nitroimidazole, in synthetic oligonucleotides and DNA treated with peroxynitrite. When calf thymus DNA or oligonucleotides were reacted with peroxynitrite at ambient temperature, the modified base 5-guanidino-4-nitroimidazole was generated along with several other products. The oligonucleotides containing the 5-guanidino-4-nitroimidazole modification were purified by reverse-phase and anion-exchange HPLC and characterized by matrix-assisted laser desorption mass spectrometry. 5-Guanidino-4-nitroimidazole formation in peroxynitrite-treated DNA was characterized after enzymatic digestion of the reacted DNA to the nucleoside level. HPLC purification and electrospray ionization mass spectrometry (with selected reaction monitoring) enabled the analysis of this modified nucleoside with high sensitivity. The yield of 5-guanidino-4-nitroimidazole formed in single-stranded DNA was approximately 10-fold higher than that found in duplex DNA. With calf thymus DNA, 5-guanidino-4-nitroimidazole was dose-dependently formed at low peroxynitrite concentrations. In stability tests, a synthetic oligonucleotide containing the 5-guanidino-4-nitroimidazole modification was only partially cleaved by hot piperidine and was a weak substrate for Fpg glycosylase repair enzyme; in addition, this site was not cleaved by endonuclease III. These results suggest that nuclear DNA containing 5-guanidino-4-nitroimidazole may not be quickly repaired by DNA repair enzyme systems. Finally, primer extension experiments revealed that this lesion is a potential DNA replication blocker when polymerization is catalyzed by polymerase alpha and polymerase I (Klenow fragment, lack of exonuclease activity) but not with human polymerase beta. Replication fidelity experiments further showed that 5-guanidino-4-nitroimidazole may cause G-->T and G-->C transversions in calf thymus polymerase alpha and E. coli polymerase I.

Animals↗
PubMed2000

Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA.

DNA mismatch repair is critical for increasing replication fidelity in organisms ranging from bacteria to humans. MutS protein, a member of the ABC ATPase superfamily, recognizes mispaired and unpaired bases in duplex DNA and initiates mismatch repair. Mutations in human MutS genes cause a predisposition to hereditary nonpolyposis colorectal cancer as well as sporadic tumours. Here we report the crystal structures of a MutS protein and a complex of MutS with a heteroduplex DNA containing an unpaired base. The structures reveal the general architecture of members of the MutS family, an induced-fit mechanism of recognition between four domains of a MutS dimer and a heteroduplex kinked at the mismatch, a composite ATPase active site composed of residues from both MutS subunits, and a transmitter region connecting the mismatch-binding and ATPase domains. The crystal structures also provide a molecular framework for understanding hereditary nonpolyposis colorectal cancer mutations and for postulating testable roles of MutS.

Adenosine Triphosphatases↗
PubMed1994

Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.

Genomic instability at simple repeated sequences (SRS) is a landmark for some sporadic and hereditary cancers of the colon. We have identified several human tumour cell lines with up to 1,000-fold increases in mutation rates for endogenous microsatellite sequences, relative to normal cells or tumour cells without the mutator phenotype and show that they are very early events in tumorigenesis. Our in vivo and in vitro results show that the genomic instability persists after transformation and that microsatellite mutations accumulate as consecutive somatic slippage events of a single or a few repeated units. This mechanism may account for the repeat expansions in triplet hereditary diseases and the same defect in replication fidelity in non-polyposis colon cancer could also contribute to the non-mendelian anticipation in these diseases.

Cell Transformation, Neoplastic↗
PubMed2026

Integrating germline and tumor sequencing to improve hereditary cancer diagnosis and care.

A subset of cancers arises due to inherited germline pathogenic variants in specific genes, known as hereditary cancers. These genes typically include tumor suppressors, DNA repair and replication fidelity genes, and occasionally oncogenes. In most hereditary cancer syndromes, Knudson's two-hit hypothesis applies, where a second somatic event inactivates the remaining allele of a tumor suppressor or DNA repair gene, leading to tumorigenesis. Advancements in genome-wide sequencing have significantly enhanced our understanding of the mutational processes involved in hereditary cancers. In particular, the assessment of microsatellite instability (MSI), tumor mutational burden (TMB), and mutational signatures has emerged as a powerful tool for the identification of hereditary tumors. Tumors with high or ultra-high TMB often reflect underlying DNA repair deficiencies, while specific mutational signatures can pinpoint the defective pathway. These tumor mutational features are especially informative in syndromes involving mismatch repair (MMR), homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and polymerase proofreading. Moreover, tumor sequencing aids in the interpretation of germline variants, identifies somatic mosaicism, and helps differentiate hereditary from sporadic cancers. Additionally, tumor molecular features associated with DNA repair deficiencies offer insights into personalized therapies, such as the use of PARP inhibitors for BRCA1/2-deficient tumors and immune checkpoint inhibitors for MMR- and polymerase proofreading-deficient cancers. Tumor profiling also uncovers actionable mutations in oncogenes like RET and VHL, which can be targeted with specific therapies. This review explores the integration of tumor molecular features with germline genetic data to refine diagnosis, risk assessment, and therapeutic strategies in hereditary cancer.

Humans↗
PubMed2005

Large area UV casting using diverse polyacrylates of microchannels separated by high aspect ratio microwalls.

Large area molding of long and deep microchannels separated by high aspect ratio microwalls is important for high sensitivity and high throughput microfluidic devices. Ultraviolet (UV) casting is a feasible, economical and convenient method of replication of such microstructures in plastics. It is shown that a wide variety of polyacrylates with diverse properties such as those made from epoxy (EP), polyurethane (UR), polyester (ES), poly (ethylene glycol) (EG) and poly(propylene glycol) (PG) can be used for the high aspect ratio (7-9) UV casting of such linear microstructures over a 100 mm diameter, enlarging the range of applications of the replicated microstructures. Some challenges arise. With the EG formulation, wavy microstructures were observed; this can be overcome by stress relaxation. With non-polar PG formulation, poor adhesion between the polyester substrate and resin can lead to delamination of the casting from the substrate during demolding; this can be overcome by pre-coating a partially cured same resin on the polyester substrate. An optimum UV irradiation time was important for cure at the deepest end of the microstructure without excessive crosslinking leading to much increased demolding forces. The viscosity and wetting capability of the formulations were found to affect replication fidelity.

Acrylates↗
PubMed2006

Physical and functional interactions between MutY glycosylase homologue (MYH) and checkpoint proteins Rad9-Rad1-Hus1.

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295-350 of hMYH and to residues 245-293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH-hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.

Active Transport, Cell Nucleus↗
PubMed2000

Escherichia coli MutS,L modulate RuvAB-dependent branch migration between diverged DNA.

This study examines the interaction between Escherichia coli MutS,L and E. coli RuvAB during E. coli RecA-promoted strand exchange. RuvAB is a branch migration complex that stimulates heterologous strand exchange. Previous studies indicate that RuvAB increases the rate at which heteroduplex products are formed by RecA, that RuvA and RuvB are required for this stimulation, and that RuvAB does not stimulate homologous strand exchange. This study indicates that MutS,L inhibit the formation of full-length heteroduplex DNA between M13-fd DNA in the presence of RuvAB, such that less than 2% of the linear substrate is converted to product. Inhibition depends on the time at which MutS,L are added to the reaction and is strongest when MutS,L are added during initiation. The kinetics of the strand exchange reaction suggest that MutS,L directly inhibit RuvAB-dependent branch migration in the absence of RecA. The inhibition requires the formation of base-base mismatches and ATP utilization; no effect on RuvAB-promoted strand exchange is seen if an ATP-deficient mutant of MutS (MutS501) is included in the reaction instead of wild-type MutS. These results are consistent with a role for MutS,L in maintaining genomic stability and replication fidelity.

Adenosine Triphosphatases↗
PubMed2003

Nickel (II) enhances benzo[a]pyrene diol epoxide-induced mutagenesis through inhibition of nucleotide excision repair in human cells: a possible mechanism for nickel (II)-induced carcinogenesis.

Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities.

The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery. To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2 Delta Bgl +1 frameshift allele. Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases delta and epsilon (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed. Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity. Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair. Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast.

Alleles↗
PubMed2002

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells.

We have measured the mutation rates of G(17) and A(17) repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)(17) repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A(17) and (CA)(17) sequences were similar in the mismatch repair proficient cells, but the mutation rate of G(17) was significantly higher than that of either A(17) or (CA)(17). In the mismatch repair deficient cells, the G(17) and (CA)(17) mutation rates were similar and were significantly higher than the A(17) rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A(17) sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G(17) sequence. These results indicate that a G(17) repeat is less stable than an A(17) repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G(17) repeats.

Animals↗
PubMed1986

Induction of acyclovir-resistant mutants of herpes simplex virus type I in athymic nude mice.

Induction of acyclovir resistance was studied in the immune compromised host by multiple passage of plaque-purified herpes simplex type I strains in athymic nude mice receiving suboptimal antiviral therapy. Mice infected with a highly pathogenic clinical isolate rapidly developed infections that were resistant to therapy. Viruses isolated from these mice had decreased in-vitro sensitivities to acyclovir, as well as altered characteristics when assayed by [125I]plaque autoradiography. In contrast, less virulent laboratory strains, or a genetically stable clinical isolate, showed no indication of mutation to resistance after extended passage in this mouse model. Highly pathogenic viruses may increase the probability of mutation to resistance because of the large amount of infectious virus they produce, while viruses of equivalent virulence may produce different amounts of drug-resistant progeny because of alterations in the replication fidelity of the viral DNA polymerase.

Acyclovir↗
PubMed1995

Mismatch repair, genetic stability and tumour avoidance.

Escherichia coli methyl-directed mismatch repair eliminates premutagenic lesions that arise via DNA biosynthetic errors; components of the repair system also block ectopic recombination between diverged DNA sequences. A mismatch-dependent, methyl-directed excision reaction that accounts for function of the system in replication fidelity has been reconstituted in a purified system dependent on ten activities. The reaction displays a broad specificity for mismatched base pairs and is characterized by an unusual bidirectional excision capability. Human cell nuclear extracts support strand-specific mismatch correction in a reaction that is similar to bacterial repair, with respect to both mismatch specificity and unusual features of mechanism. Like the bacterial system, the human pathway also functions in mutation avoidance because several classes of mutator human cells are deficient in the reaction. These include an alkylation-tolerance cell line that is resistant to the cytotoxic action of N-methyl-N'-nitro-nitrosoguanidine, as well as hypermutable RER+ tumour cells such as those associated with hereditary non-polyposis colon cancer. In vitro experiments indicate that the human repair reaction is dependent on at least six activities, excluding DNA ligase, and that distinct defects in the system can lead to the RER+ phenotype.

DNA Repair↗
PubMed2007

The emergence of antibiotic resistance by mutation.

The emergence of mutations in nucleic acids is one of the major factors underlying evolution, providing the working material for natural selection. Most bacteria are haploid for the vast majority of their genes and, coupled with typically short generation times, this allows mutations to emerge and accumulate rapidly, and to effect significant phenotypic changes in what is perceived to be real-time. Not least among these phenotypic changes are those associated with antibiotic resistance. Mechanisms of horizontal gene spread among bacterial strains or species are often considered to be the main mediators of antibiotic resistance. However, mutational resistance has been invaluable in studies of bacterial genetics, and also has primary clinical importance in certain bacterial species, such as Mycobacterium tuberculosis and Helicobacter pylori, or when considering resistance to particular antibiotics, especially to synthetic agents such as fluoroquinolones and oxazolidinones. In addition, mutation is essential for the continued evolution of acquired resistance genes and has, e.g., given rise to over 100 variants of the TEM family of beta-lactamases. Hypermutator strains of bacteria, which have mutations in genes affecting DNA repair and replication fidelity, have elevated mutation rates. Mutational resistance emerges de novo more readily in these hypermutable strains, and they also provide a suitable host background for the evolution of acquired resistance genes in vitro. In the clinical setting, hypermutator strains of Pseudomonas aeruginosa have been isolated from the lungs of cystic fibrosis patients, but a more general role for hypermutators in the emergence of clinically relevant antibiotic resistance in a wider variety of bacterial pathogens has not yet been proven.

Anti-Bacterial Agents↗
PubMed2006

Physical and functional interactions between Escherichia coli MutY glycosylase and mismatch repair protein MutS.

Escherichia coli MutY and MutS increase replication fidelity by removing adenines that were misincorporated opposite 7,8-dihydro-8-oxo-deoxyguanines (8-oxoG), G, or C. MutY DNA glycosylase removes adenines from these mismatches through a short-patch base excision repair pathway and thus prevents G:C-to-T:A and A:T-to-G:C mutations. MutS binds to the mismatches and initiates the long-patch mismatch repair on daughter DNA strands. We have previously reported that the human MutY homolog (hMYH) physically and functionally interacts with the human MutS homolog, hMutSalpha (Y. Gu et al., J. Biol. Chem. 277:11135-11142, 2002). Here, we show that a similar relationship between MutY and MutS exists in E. coli. The interaction of MutY and MutS involves the Fe-S domain of MutY and the ATPase domain of MutS. MutS, in eightfold molar excess over MutY, can enhance the binding activity of MutY with an A/8-oxoG mismatch by eightfold. The MutY expression level and activity in mutS mutant strains are sixfold and twofold greater, respectively, than those for the wild-type cells. The frequency of A:T-to-G:C mutations is reduced by two- to threefold in a mutS mutY mutant compared to a mutS mutant. Our results suggest that MutY base excision repair and mismatch repair defend against the mutagenic effect of 8-oxoG lesions in a cooperative manner.

Binding Sites↗
PubMed1990

The single-stranded DNA-binding protein of Escherichia coli.

The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.

Amino Acid Sequence↗
PubMed2002

Guanine and 7,8-dihydro-8-oxo-guanine-specific oxidation in DNA by chromium(V).

The hexavalent oxidation state of chromium [Cr(VI)] is a well-established human carcinogen, although the mechanism of cancer induction is currently unknown. Intracellular reduction of Cr(VI) forms Cr(V), which is thought to play a fundamental role in the mechanism of DNA damage by this carcinogen. Two separate pathways of DNA damage, an oxidative pathway and a metal-binding pathway, have been proposed to account for the lesions observed in cell systems. We have used a model Cr(V) complex, N,N-ethylenebis(salicylidene-animato)oxochromium(V) [Cr(V)-Salen], to investigate the oxidative pathway of DNA damage and to elucidate the lesions generated from this oxidation process. Reaction of Cr(V)-Salen with synthetic oligonucleotides produced guanine-specific lesions that were not 8-oxo-2'-deoxyguanosine, based on the inability of iridium(IV) to further oxidize these sites. Oxidation products were identified using a 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-G) containing oligonucleotide to increase the yields of product for identification by electrospray ionization mass spectrometry. The guanine-based lesions observed by mass spectrometry corresponded to the lesions guanidinohydantoin and spiroiminodihydantoin. The effects of these Cr(V)-Salen-induced lesions on DNA replication fidelity was assayed using a polymerase-based misincorporation assay. These lesions produced G --> T transversion mutations and polymerase stops at levels greater than those observed for 8-oxo-G. These data suggest a model by which chromate can cause DNA damage leading to mutations and cancer.

Carcinogens, Environmental↗
PubMed2005

Conditional coalescent trees with two mutation rates and their application to genomic instability.

Humans have invested several genes in DNA repair and fidelity replication. To account for the disparity between the rarity of mutations in normal cells and the large number of mutations present in cancer, an hypothesis is that cancer cells must exhibit a mutator phenotype (genomic instability) during tumor progression, with the initiation of abnormal mutation rates caused by the loss of mismatch repair. In this study we introduce a stochastic model of mutation in tumor cells with the aim of estimating the amount of genomic instability due to the alteration of DNA repair genes. Our approach took into account the difficulties generated by sampling within tumoral clones and the fact that these clones must be difficult to isolate. We provide corrections to two classical statistics to obtain unbiased estimators of the raised mutation rate, and we show that large statistical errors may be associated with such estimators. The power of these new statistics to reject genomic instability is assessed and proved to increase with the intensity of mutation rates. In addition, we show that genomic instability cannot be detected unless the raised mutation rates exceed the normal rates by a factor of at least 1000.

Computational Biology↗
PubMed1992

The effects of dNTP pool imbalances on frameshift fidelity during DNA replication.

The use of unequal concentrations of the four deoxynucleoside triphosphates (dNTPs) in DNA polymerization reactions alters base substitution error rates in a predictable way. Less is known about the effects of substrate imbalances on base addition and deletion error rates. Thus, we examined pool bias effects on frameshift fidelity during DNA synthesis catalyzed by replicative DNA polymerases. Imbalanced pools altered the frameshift fidelity of the human immunodeficiency virus type-1 reverse transcriptase. Both mutagenic and antimutagenic effects were observed for minus-one, plus-one, and minus-two nucleotide errors, in a highly sequence-specific manner. Most of this specificity can be rationalized by either of two models. One involves frameshifts initiated by pool bias-induced nucleotide misinsertion, and the other involves pool bias-initiated template-primer slippage. Several examples of complex mutations were also recovered more than once in small mutant collections. These contained closely spaced single-base substitution and minus-one base frameshift changes. The two changes occurred at a frequency much higher than predicted if they were generated independently. This suggests that when the polymerase makes one mistake, the probability that it will make a second mistake within the next few incorporations increases significantly. Perturbation of dNTP pools also affected the frameshift fidelity of the replicative yeast DNA polymerase alpha. In reactions containing a low concentration of one dNTP, the error rate increased for one-nucleotide deletions at homopolymeric template nucleotides complementary to the dNTP whose concentration was low. We extended this approach to determine the frameshift fidelity of simian virus 40 origin-dependent semiconservative replication of double-stranded DNA in extracts of human cells. In reactions performed with an equal concentration of all four dNTPs, replication was highly accurate for minus-one-nucleotide errors. However, when the concentration of one dNTP was decreased, the replication error rate increased at complementary, homopolymeric template positions. This response provides an approach for describing frameshift accuracy during replication of the leading and lagging strands.

Base Sequence↗