Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “replication fidelity”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 361 records · Page 20Linked to original sources
PubMed1994

Roadblocks and detours during DNA replication: mechanisms of mutagenesis in mammalian cells.

Mutations in specific genes result in birth defects, cancer, inherited diseases or lethality. The frequency with which DNA damage is converted to mutations increases dramatically when the cellular genome is replicated. Although DNA damage poses special problems to the fidelity of DNA replication, efficient mechanisms exist in mammalian cells which function to replicate their genome despite the presence of many damaged sites. These mechanisms operate in either error-prone or error-free modes of DNA synthesis, and frequently involve DNA strand-pairing reactions. Genetic studies in yeast and other eukaryotes suggest that replication through DNA damage is highly regulated and catalysed by complex biochemical machineries composed of many specialized gene products. Knowledge of the molecular details by which such factors facilitate the replication of damaged DNA in mammalian cells should reveal basic rules about how DNA damage induces mutagenesis and carcinogenesis.

Animals↗
PubMed1991

Mammalian DNA replication: mutation biases and the mutation rate.

Experimental studies have shown that the fidelity of DNA replication can be affected by the concentrations of free deoxyribonucleotides present in the cell. Replication of mammalian chromosomes is achieved using pools of newly-synthesized deoxyribonucleotides which fluctuate during the cell cycle. Since regions of mammalian chromosomes are replicated sequentially, there is the potential for differences among mammalian loci in both the relative and absolute frequencies of the various transitional and transversional mutations which may occur. Where these mutations are effectively neutral, at silent sites in genes and in non-coding sequences, this may result in different rates of evolution and in different base compositions, as have been observed in data from mammalian genes. A simple model of the DNA replication process is developed to describe how the mutation rate could be affected by the G + C contents of the deoxyribonucleotide pools and of the replicating DNA. Mutation rates are predicted to vary from locus to locus; only in the particular case of identical G + C contents in the DNA locus and the deoxyribonucleotide pools, and no proofreading, will the mutation rate be uniform over all loci.

Animals↗
PubMed1989

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

Amino Acid Sequence↗
PubMed2004

Escherichia coli DNA polymerase I (Klenow fragment) uses a hydrogen-bonding fork from Arg668 to the primer terminus and incoming deoxynucleotide triphosphate to catalyze DNA replication.

Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1 'R,3 'R,4 'R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter kpol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in kpol. These results are consistent with a hydrogen-bonding fork from Arg668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.

Arginine↗
PubMed2002

UV damage, DNA repair and skin carcinogenesis.

Skin cancer is unique among human cancers in its etiology, accessibility and the volume of detailed knowledge now assembled concerning its molecular mechanisms of origin. The major carcinogenic agent for most skin cancers is well established as solar ultraviolet light. This is absorbed in DNA with the formation of UV-specific dipyrimidine photoproducts. These can be repaired by nucleotide excision repair or replicated by low fidelity class Y polymerases. Insufficient repair followed by errors in replication produce characteristic mutations in dipyrimidine sequences that may represent initiation events in carcinogenesis. Chronic exposure to UVB results in disruption of the epithelial structure and expansion of pre-malignant clones which undergo further genomic changes leading to full malignancy. Genetic diseases in DNA repair, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, show varied elevated symptoms of sun sensitivity involving skin cancers and other symptoms including neurological degeneration and developmental delays. In humans, only xeroderma pigmentosum shows high levels of cancer, but mouse strains, with any of the genes corresponding to these diseases knocked-out, show elevated skin carcinogenesis. The three major skin cancers exhibit characteristic molecular changes defined by certain genes and associated pathways. Squamous cell carcinoma involves mutations in the p53 gene; basal cell carcinoma involves mutations in the PATCHED gene, and melanoma in the p16 gene. The subsequent development of malignant tumors involves many additional genomic changes that have yet to be fully cataloged.

Animals↗
PubMed2003

Mutations in DNA polymerase gamma cause error prone DNA synthesis in human mitochondrial disorders.

This paper summarizes recent advances in understanding the links between the cell's ability to maintain integrity of its mitochondrial genome and mitochondrial genetic diseases. Human mitochondrial DNA is replicated by the two-subunit DNA polymerase gamma (polgamma). We investigated the fidelity of DNA replication by polgamma with and without exonucleolytic proofreading and its p55 accessory subunit. Polgamma has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides. Progressive external ophthalmoplegia (PEO) is a rare disease characterized by the accumulation of large deletions in mitochondrial DNA. Recently, several mutations in the polymerase and exonuclease domains of the human polgamma have been shown to be associated with PEO. We are analyzing the effect of these mutations on the human polgamma enzyme. In particular, three autosomal dominant mutations alter amino acids located within polymerase motif B of polgamma. These residues are highly conserved among family A DNA polymerases, which include T7 DNA polymerase and E.coli pol I. These PEO mutations have been generated in polgamma to analyze their effects on overall polymerase function as well as the effects on the fidelity of DNA synthesis. One mutation in particular, Y955C, was found in several families throughout Europe, including one Belgian family and five unrelated Italian families. The Y955C mutant polgamma retains a wild-type catalytic rate but suffers a 45-fold decrease in apparent binding affinity for the incoming dNTP. The Y955C derivative is also much less accurate than is wild-type polgamma, with error rates for certain mismatches elevated by 10- to 100-fold. The error prone DNA synthesis observed for the Y955C polgamma is consistent with the accumulation of mtDNA mutations in patients with PEO. The effects of other polgamma mutations associated with PEO are discussed.

Amino Acid Sequence↗
PubMed2005

Probing the active site tightness of DNA polymerase in subangstrom increments.

We describe the use of a series of gradually expanded thymine nucleobase analogs in probing steric effects in DNA polymerase efficiency and fidelity. In these nonpolar compounds, the base size was increased incrementally over a 1.0-A range by use of variably sized atoms (H, F, Cl, Br, and I) to replace the oxygen molecules of thymine. Kinetics studies with DNA Pol I (Klenow fragment, exonuclease-deficient) in vitro showed that replication efficiency opposite adenine increased through the series, reaching a peak at the chlorinated compound. Efficiency then dropped markedly as a steric tightness limit was apparently reached. Importantly, fidelity also followed this trend, with the fidelity maximum at dichlorotoluene, the largest compound that fits without apparent repulsion. The fidelity at this point approached that of wild-type thymine. Surprisingly, the maximum fidelity and efficiency was found at a base pair size significantly larger than the natural size. Parallel bypass and mutagenesis experiments were then carried out in vivo with a bacterial assay for replication. The cellular results were virtually the same as those seen in solution. The results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity. In addition, the results suggest that even high-fidelity replicative enzymes have more steric room than necessary, possibly to allow for an evolutionarily advantageous mutation rate.

Base Pairing↗
PubMed1999

DNA structure and polymerase fidelity.

The accuracy of DNA replication results from both the intrinsic DNA polymerase fidelity and the DNA sequence. Although the recent structural studies on polymerases have brought new insights on polymerase fidelity, the role of DNA sequence and structure is less well understood. Here, the analysis of the crystal structures of hotspots for polymerase slippage including (CA)n and (A)n tracts in different intermolecular contexts reveals that, in the B-form, these sequences share common structural alterations which may explain the high rate of replication errors. In particular, a two-faced "Janus-like" structure with shifted base-pairs in the major groove but an apparent normal geometry in the minor groove constitutes a molecular decoy specifically suitable to mislead the polymerases. A model of the rat polymerase beta bound to this structure suggests that an altered conformation of the nascent template-primer duplex can interfere with correct nucleotide incorporation by affecting the geometry of the active site and breaking the rules of base-pairing, while at the same time escaping enzymatic mechanisms of error discrimination which scan for the correct geometry of the minor groove.In contrast, by showing that the A-form greatly attenuates the sequence-dependent structural alterations in hotspots, this study suggests that the A-conformation of the nascent template-primer duplex at the vicinity of the polymerase active site will contribute to fidelity. The A-form may play the role of a structural buffer which preserves the correct geometry of the active site for all sequences. The detailed comparison of the conformation of the nascent template-primer duplex in the available crystal structures of DNA polymerase-DNA complexes shows that polymerase beta, the least accurate enzyme, is unique in binding to a B-DNA duplex even close to its active site. This model leads to several predictions which are discussed in the light of published experimental data.

Animals↗
PubMed1986

Capacity of RecA protein to bind preferentially to UV lesions and inhibit the editing subunit (epsilon) of DNA polymerase III: a possible mechanism for SOS-induced targeted mutagenesis.

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. Most SOS-induced mutations probably occur during replication across a DNA lesion (targeted mutagenesis). We have suggested previously that RecA might participate in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g., pyrimidine dimer) because of its partially unwound character; DNA polymerase III (polIII) will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site with reduced fidelity. In this report, we analyze at a biochemical level two major predictions of this model. With respect to lesion recognition, we show that purified RecA protein binds more efficiently to UV-irradiated double-stranded DNA than to nonirradiated DNA, as judged by filter-binding and gel electrophoresis assays. With respect to replication fidelity, Fersht and Knill-Jones [Fersht, A. R. & Knill-Jones, J. W. (1983) J. Mol. Biol. 165, 669-682] have found that RecA inhibits the 3'----5' exonuclease (editing function) of polIII holoenzyme. We extend this observation by demonstrating that RecA inhibits the exonuclease of the purified editing subunit of polIII, epsilon protein. Thus, we suggest that the activities of RecA required for targeted mutagenesis are lesion-recognition, followed by localized inhibition of the editing capacity of the epsilon subunit of polIII holoenzme. In this proposed mechanism, one activation signal for RecA for mutagenesis is the lesion itself. Because UV-irradiated, double-stranded DNA efficiently activates RecA for cleavage of the LexA repressor, the lesion itself may also often serve as an activation signal for induction of SOS-controlled genes.

Antibody Diversity↗
PubMed2002

Noisy clues to the origin of life.

The origin of stable self-replicating molecules represents a fundamental obstacle to the origin of life. The low fidelity of primordial replicators places restrictions on the quantity of information encoded in a primitive nucleic acid alphabet. Further difficulties for the origin of life are the role of drift in small primordial populations, reducing the rate of fixation of superior replicators, and the hostile conditions increasing developmental noise. Thus, mutation, noise and drift are three different stochastic effects that are assumed to make the evolution of life improbable. Here we show, to the contrary, how noise present in hostile early environments can increase the probability of faithful replication, by amplifying selection in finite populations. Noise has negative consequences in infinite populations, whereas in finite populations, we observe a synergistic interaction among noise sources. Hence, two factors formerly considered inimical to the origin of life-developmental noise and drift in small populations-can in combination give rise to conditions favourable to robust replication.

Biological Evolution↗
PubMed2015

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Animals↗
PubMed2004

Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase.

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.

Amines↗
PubMed2002

The roles of REV3 and RAD57 in double-strand-break-repair-induced mutagenesis of Saccharomyces cerevisiae.

The DNA synthesis associated with recombinational repair of chromosomal double-strand breaks (DSBs) has a lower fidelity than normal replicative DNA synthesis. Here, we use an inverted-repeat substrate to monitor the fidelity of repair of a site-specific DSB. DSB induction made by the HO endonuclease stimulates recombination >5000-fold and is associated with a >1000-fold increase in mutagenesis of an adjacent gene. We demonstrate that most break-repair-induced mutations (BRIMs) are point mutations and have a higher proportion of frameshifts than do spontaneous mutations of the same substrate. Although the REV3 translesion DNA polymerase is not required for recombination, it introduces approximately 75% of the BRIMs and approximately 90% of the base substitution mutations. Recombinational repair of the DSB is strongly dependent upon genes of the RAD52 epistasis group; however, the residual recombinants present in rad57 mutants are associated with a 5- to 20-fold increase in BRIMs. The spectrum of mutations in rad57 mutants is similar to that seen in the wild-type strain and is similarly affected by REV3. We also find that REV3 is required for the repair of MMS-induced lesions when recombinational repair is compromised. Our data suggest that Rad55p/Rad57p help limit the generation of substrates that require pol zeta during recombination.

Adenosine Triphosphatases↗
PubMed2004

Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase.

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.

Base Pairing↗
PubMed2005

DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli.

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair-defective strains containing the polBex1 allele--encoding a polymerase-proficient but exonucleolytically defective Pol II--displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading- versus lagging-strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE-produced misinsertion errors. A second role of Pol II is to protect mismatched 3' termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.

Alleles↗
PubMed2006

Characterization of Escherichia coli translesion synthesis polymerases and their accessory factors.

Members of the Y family of DNA polymerases are specialized to replicate lesion-containing DNA. However, they lack 3'-5' exonuclease activity and have reduced fidelity compared to replicative polymerases when copying undamaged templates, and thus are potentially mutagenic. Y family polymerases must be tightly regulated to prevent aberrant mutations on undamaged DNA while permitting replication only under conditions of DNA damage. These polymerases provide a mechanism of DNA damage tolerance, confer cellular resistance to a variety of DNA-damaging agents, and have been implicated in bacterial persistence. The Y family polymerases are represented in all domains of life. Escherichia coli possesses two members of the Y family, DNA pol IV (DinB) and DNA pol V (UmuD'(2)C), and several regulatory factors, including those encoded by the umuD gene that influence the activity of UmuC. This chapter outlines procedures for in vivo and in vitro analysis of these proteins. Study of the E. coli Y family polymerases and their accessory factors is important for understanding the broad principles of DNA damage tolerance and mechanisms of mutagenesis throughout evolution. Furthermore, study of these enzymes and their role in stress-induced mutagenesis may also give insight into a variety of phenomena, including the growing problem of bacterial antibiotic resistance.

DNA↗
PubMed2006

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (alpha, oligomeric state unknown) and a small subunit R2 (beta(2)). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe)(2)-Y.] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (betabeta'), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, betabeta' is redistributed from the nucleus to the cytoplasm in a checkpoint-dependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in betabeta' nuclear localization: the importin beta homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of betabeta' nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with betabeta'. Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of betabeta' by Kap122 or as an anchor to retain betabeta' in the nucleus.

Catalysis↗