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At least 289 records · Page 16Linked to original sources
PubMed1989

Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins.

We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.

Animals↗
PubMed2003

Yeast DNA polymerase eta makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate.

DNA polymerase eta (Pol eta) functions in the proficient bypass of a variety of DNA lesions. Relative to the replicative polymerases, Pol eta has a greater tolerance for distorted DNA geometries and possesses a low fidelity. X-ray crystal structures and studies with nucleotide analogs have implicated interactions with the DNA minor groove as being crucial for the high fidelity of replicative DNA polymerases. To determine whether Pol eta also makes such functionally important contacts with the DNA minor groove, here we examine the effects on Pol eta-catalyzed nucleotide incorporation when 3-deazaguanine, a base analog that lacks the ability to form minor-groove hydrogen bonds with the protein, is substituted for guanine at various positions in the DNA. From these studies, we conclude that Pol eta makes only a single functional contact with the DNA minor groove at the position of the incoming nucleotide; in this regard, Pol eta differs from high-fidelity DNA polymerases that are unable to replicate through DNA lesions. These results help explain the proficient ability of Pol eta for bypassing distorting DNA lesions.

Base Sequence↗
PubMed1992

HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.

To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined. An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis. Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold. Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template. Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene. Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex.

Bacterial Proteins↗
PubMed2003

Constitutive and regulated expression of the mouse Dinb (Polkappa) gene encoding DNA polymerase kappa.

A recently discovered group of novel polymerases are characterized by significantly reduced fidelity of DNA synthesis in vitro. This feature is consistent with the relaxed fidelity required for the replicative bypass of various types of base damage that frequently block high fidelity replicative polymerases. The present studies demonstrate that the specialized DNA polymerase kappa (polkappa) is uniquely and preferentially expressed in the adrenal cortex and testis of the mouse, as well as in a variety of other tissues. The adrenal cortex is the sole site of detectable expression of the Polkappa gene in mouse embryos. This adrenal expression pattern is consistent with a requirement for polkappa for the replicative bypass of DNA base damage generated during steroid biosynthesis. The expression pattern of polkappa in the testis is specific for particular stages of spermatogenesis and is distinct from the expression pattern of several other low fidelity DNA polymerases that are also expressed during spermatogenesis. The mouse (but not the human) Polkappa gene is primarily regulated by the p53 gene and is upregulated in response to exposure to various DNA-damaging agents in a p53-dependent manner.

Adrenal Cortex↗
PubMed1996

Structure of the UmuD' protein and its regulation in response to DNA damage.

For life to be sustained, mistakes in DNA repair must be tolerated when damage obscures the genetic information. In bacteria such as Escherichia coli, DNA damage elicits the well regulated 'SOS response'. For the extreme case of damage that cannot be repaired by conventional enzymes, there are proteins that allow the replication of DNA through such lesions, but with a reduction in the fidelity of replication. Essential proteins in this mutagenic process are RecA, DNA polymerase III, UmuD, UmuD' and UmuC (umu: UV mutagenesis). Regulation of this response involves a RecA-mediated self-cleavage of UmuD to produce UmuD'. To understand this system in more detail, we have determined the crystal structure of the E. coli UmuD' mutagenesis protein at 2.5 A resolution. Globular heads folded in an unusual Beta-structure associate to form molecular dimers, and extended amino-terminal tails associate to produce crystallized filaments. The structure provides insight into the mechanism of the self-cleavage reaction that UmuD-like proteins undergo as part of the global SOS response.

Amino Acid Sequence↗
PubMed2003

Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways.

The fidelity of DNA replication and repair processes is critical for maintenance of genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production and thus plays an essential role in DNA synthesis. The level and activity of RNR are highly regulated by the cell cycle and DNA damage checkpoints, which maintain optimal dNTP pools required for genetic fidelity. RNRs are composed of a large subunit that binds the nucleoside diphosphate substrates and allosteric effectors and a small subunit that houses the di-iron tyrosyl radical cofactor essential for the reduction process. In Saccharomyces cerevisiae, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). Here we report the subcellular localization of Rnr1-4 during normal cell growth and the redistribution of Rnr2 and Rnr4 in response to DNA damage and replicational stress. During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm and Rnr2 and Rnr4 are predominantly present in the nucleus. Under genotoxic stress, Rnr2 and Rnr4 become redistributed to the cytoplasm in a checkpoint-dependent manner. Subcellular redistribution of Rnr2 and Rnr4 can occur in the absence of the transcriptional induction of the RNR genes after DNA damage and likely represents a posttranslational event. These results suggest a mechanism by which DNA damage checkpoint modulates RNR activity through the temporal and spatial regulation of its subunits.

DNA Damage↗
PubMed1995

Replication error rates for G.dGTP, T.dGTP, and A.dGTP mispairs and evidence for differential proofreading by leading and lagging strand DNA replication complexes in human cells.

We have determined the fidelity of DNA replication by human cell extracts in reactions containing excess dGTP. Replication errors were scored using two M13 DNA substrates having the replication origin on opposite sides of the lacZ alpha-complementation gene. The data suggest that the average rates for replication errors resulting from G(template), T.dGTP, and A.dGTP mispairs are 25 x 10(-6), 12 x 10(-6), and 3 x 10(-6), respectively. The data also suggest that error rates for both the (+) and (-) strands differ by less than 2-fold when they are replicated either as the leading or lagging strand. This is in contrast to the 33- and 8-fold differences observed earlier for G.dTTP and C.dTTP mispairs on the (+) strand when replicated by the leading or lagging strand complex (Roberts, J. D., Izuta, S., Thomas, D. C., and Kunkel, T. A. (1994) J. Biol. Chem. 269, 1711-1717). Thus, the relative fidelity of the leading and lagging strand replication proteins varies with the mispair and sequence considered. Misincorporation of dGTP preferentially occurs at template positions where dGTP is the next correct nucleotide to be incorporated. This "next nucleotide" effect is characteristic of reduced exonucleolytic proofreading and suggests that these replication errors are normally proofread efficiently. Fidelity measurements performed in the absence or presence of dGMP, an inhibitor of proofreading exonuclease activity, suggest that the leading strand replication complex proofreads some mispairs more efficiently than does the lagging strand replication complex.

Base Sequence↗
PubMed1983

Paper replication method for isolation of radiation-sensitive mutants.

A filter paper replication system particularly useful for isolation of radiation-sensitive mutants of pigmented bacteria was devised. The fidelity of replication was high. Adhesion between a paper disk and a properly dried master plate provided adequate contact pressure. The replicas arising from this technique constitute a convenient apparatus for general application in isolation of clones sensitive to a discriminating treatment.

Bacteriological Techniques↗
PubMed1996

Exonucleolytic proofreading during replication of repetitive DNA.

We are attempting to understand the processes required to accurately replicate the repetitive DNA sequences whose instability is associated with several human diseases. Here we test the hypothesis that the contribution of exonucleolytic proofreading to frameshift fidelity during replication of repetitive DNA sequences diminishes as the number of repeats in the sequence increases. The error rates of proofreading-proficient T7, T4, and Pyrococcus furiosis DNA polymerases are compared to their exonuclease-deficient derivatives, for +1 and -1 base errors in homopolymeric repeat sequences of three to eight base pairs. All three exonuclease-deficient polymerases produce frameshift errors during synthesis at rates that increase as a function of run length, suggesting the involvement of misaligned intermediates. Their wild-type counterparts are all much more accurate, suggesting that the majority of the intermediates are corrected by proofreading. However, the contribution of the exonuclease to fidelity decreases substantially as the length of the homopolymeric run increases. For example, the exonuclease enhances the frameshift fidelity of T7 DNA polymerase in a run of three A.T base pairs by 160-fold, similar to its contribution to base substitution fidelity. However, in a run of eight consecutive A.T base pairs, the exonuclease only enhances frameshift fidelity by 7-fold. A similar pattern was observed with T4 and Pfu DNA polymerases. Thus, both polymerase selectivity and exonucleolytic proofreading efficiency are diminished during replication of repetitive sequences. This may place an increased relative burden on post-replication repair processes to reduce rates of addition and deletion mutations in organisms whose genome contains abundant simple repeat DNA sequences.

Base Sequence↗
PubMed2003

Fidelity of the methylation pattern and its variation in the genome.

The methylated or unmethylated status of a CpG site is copied faithfully from parental DNA to daughter DNA, and functions as a cellular memory. However, no information is available for the fidelity of methylation pattern in unmethylated CpG islands (CGIs) or its variation in the genome. Here, we determined the methylation status of each CpG site on each DNA molecule obtained from clonal populations of normal human mammary epithelial cells. Methylation pattern error rates (MPERs) were calculated based upon the deviation from the methylation patterns that should be obtained if the cells had 100% fidelity in replicating the methylation pattern. Unmethylated CGIs in the promoter regions of five genes showed MPERs of 0.018-0.032 errors/site/21.6 generations, and the fidelity of methylation pattern was calculated as 99.85%-99.92%/site/generation. In contrast, unmethylated CGIs outside the promoter regions showed MPERs more than twice as high (P < 0.01). Methylated regions, including a CGI in the MAGE-A3 promoter and DMR of the H19 gene, showed much lower MPERs than unmethylated CGIs. These showed that errors in methylation pattern were mainly due to de novo methylations in unmethylated regions. The differential MPERs even among unmethylated CGIs indicated that a promoter-specific protection mechanism(s) from de novo methylation was present.

Base Composition↗
PubMed2004

Structure of a high fidelity DNA polymerase bound to a benzo[a]pyrene adduct that blocks replication.

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Benzo(a)pyrene↗
PubMed1994

The fidelity of the human leading and lagging strand DNA replication apparatus with 8-oxodeoxyguanosine triphosphate.

A product of oxidative metabolism, 8-oxodeoxyguanosine triphosphate (8-O-dGTP), readily pairs with adenine during DNA replication, ultimately causing A.T-->C.G transversions. This study utilized 8-O-dGTP as a probe to examine the fidelity of the leading and lagging strand replication apparatus in extracts of HeLa cells. Simian virus (SV) 40 T antigen-dependent DNA replication reactions were performed with two M13mp2 vectors with the SV40 origin located on opposite sides of the lacZ alpha sequence used to score replication errors. The presence of 8-O-dGTP at equimolar concentration with each of the 4 normal dNTPs resulted in a > 46-fold increase in error rate for A.T-->C.G transversion over that observed in the absence of 8-O-dGTP. A similar average error rate was observed on the (+) and (-) strands in both vectors, suggesting that the fidelity of replication by leading and lagging strand replication proteins is similar for the dA.8-O-dGMP mispair. Replication fidelity in the presence of 8-O-dGTP was reduced on both strands when an inhibitor of exonucleolytic proofreading (dGMP) was added to the reaction. These data suggest that the majority of dA.8-O-dGMP mispairs are proofread by both leading and lagging strand replication proteins.

Base Sequence↗
PubMed1991

Basal body movements as a mechanism for mitochondrial genome segregation in the trypanosome cell cycle.

The mitochondrial genome of Trypanosoma brucei is organized in the form of a complex catenated network of circular DNA molecules. This mass of DNA, known as the kinetoplast, is present at a unique site in the single mitochondrion, and is replicated in a discrete, periodic S phase of the cell cycle. The single-copy nature of the kinetoplast suggests that there is a mechanism ensuring segregation fidelity of replicated copies to each daughter cell. Historically, speculation regarding the nature of this mechanism has often attributed significance to the close association between the kinetoplast and the flagellum basal body. We provide here direct evidence that this mitochondrial DNA complex is indeed linked to the basal body, and segregation of the kinetoplast DNA is dependent on a microtubule-mediated separation of the new and old flagellar basal bodies during the cell cycle. This unique system may represent the remnants of an evolutionarily archaic mechanism for genome segregation.

Animals↗
PubMed2003

Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast.

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.

Base Pair Mismatch↗
PubMedDate not supplied

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins.

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.

Animals↗
PubMed2005

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells.

With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly(l-lysine)-g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL-g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.

Adsorption↗
PubMed1999

A specific partner for abasic damage in DNA.

In most models of DNA replication, Watson-Crick hydrogen bonding drives the incorporation of nucleotides into the new strand of DNA and maintains the complementarity of bases with the template strand. Studies with nonpolar analogues of thymine and adenine, however, have shown that replication is still efficient in the absence of hydrogen bonds. The replication of base pairs might also be influenced by steric exclusion, whereby inserted nucleotides need to be the correct size and shape to fit the active site against a template base. A simple steric-exclusion model may not require Watson-Crick hydrogen bonding to explain the fidelity of replication, nor should canonical purine and pyrimidine shapes be necessary for enzymatic synthesis of a base pair if each can fit into the DNA double helix without steric strain. Here we test this idea by using a pyrene nucleoside triphosphate (dPTP) in which the fluorescent 'base' is nearly as large as an entire Watson-Crick base pair. We show that the non-hydrogen-bonding dPTP is efficiently and specifically inserted by DNA polymerases opposite sites that lack DNA bases. The efficiency of this process approaches that of a natural base pair and the specificity is 10(2)-10(4)-fold. We use these properties to sequence abasic lesions in DNA, which are a common form of DNA damage in vivo. In addition to their application in identifying such genetic lesions, our results show that neither hydrogen bonds nor purine and pyrimidine structures are required to form a base pair with high efficiency and selectivity. These findings confirm that steric complementarity is an important factor in the fidelity of DNA synthesis.

Base Pairing↗
PubMedDate not supplied

Structure of an adenine-cytosine base pair in DNA and its implications for mismatch repair.

Mutational pathways rely on introducing changes in the DNA double helix. This may be achieved by the incorporation of a noncomplementary base on replication or during genetic recombination, leading to substitution mutation. In vivo studies have shown that most combinations of base-pair mismatches can be accommodated in the DNA double helix, albeit with varying efficiencies. Fidelity of replication requires the recognition and excision of mismatched bases by proofreading enzymes and post-replicative mismatch repair systems. Rates of excision vary with the type of mismatch and there is some evidence that these are influenced by the nature of the neighbouring sequences. However, there is little experimental information about the molecular structure of mismatches and their effect on the DNA double helix. We have recently determined the crystal structures of several DNA fragments with guanine X thymine and adenine X guanine mismatches in a full turn of a B-DNA helix and now report the nature of the base pairing between adenine and cytosine in an isomorphous fragment. The base pair found in the present study is novel and we believe has not previously been demonstrated. Our results suggest that the enzymatic recognition of mismatches is likely to occur at the level of the base pairs and that the efficiency of repair can be correlated with structural features.

Adenine↗