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PubMed2003

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

BACKGROUND: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. METHODS: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. CONCLUSIONS: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

Gene Amplification↗
PubMed2004

Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells.

BACKGROUND: cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells. RESULTS: We performed parallel experiments on identical samples using a dye-swap design with ANOVA and an experimental design that excludes systematic biases by "correcting" experimental/control hybridization ratios with control/control hybridizations on a spot-by-spot basis. We refer to this approach as the "control correction method" (CCM). Using replicate arrays, we identified a decrease in proliferation genes and an increase in differentiation genes. Using an arbitrary cut-off of 1.7-fold and p values <0.05, we identified a total of 32 differentially expressed genes, 9 with the dye-swap method, 18 with the CCM, and 5 genes with both methods. 23 of these 32 genes were subsequently verified by northern blotting. Most of these were <2-fold changes. While the dye-swap method (using either ANOVA or Bayesian analysis) detected a smaller number of genes (14-16) compared to the CCM (46), it was more accurate (89-92% vs. 75%). Compared to the northern blot results, for most genes, the microarray results underestimated the fold change, implicating the importance of detecting these small changes. CONCLUSIONS: We validated two experimental design paradigms for cDNA microarray experiments capable of detecting small (<2-fold) changes in gene expression with excellent fidelity that revealed potentially important genes associated with the anti-proliferative effects of neuregulin on MCF10AT breast epithelial cells.

Blotting, Northern↗
PubMed2006

Correct and incorrect nucleotide incorporation pathways in DNA polymerase beta.

Tracking the structural and energetic changes in the pathways of DNA replication and repair is central to the understanding of these important processes. Here we report favorable mechanisms of the polymerase-catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA by using a novel protocol involving energy minimizations, dynamics simulations, quasi-harmonic free energy calculations, and mixed quantum mechanics/molecular mechanics dynamics simulations. Though the pathway proposed may not be unique and invites variations, geometric and energetic arguments support the series of transient intermediates in the phosphoryl transfer pathways uncovered here for both the G:C and G:A systems involving a Grotthuss hopping mechanism of proton transfer between water molecules and the three conserved aspartate residues in pol beta's active-site. In the G:C system, the rate-limiting step is the initial proton hop with a free energy of activation of at least 17 kcal/mol, which corresponds closely to measured k(pol) values. Fidelity discrimination in pol beta can be explained by a significant loss of stability of the closed ternary complex of the enzyme in the G:A system and much higher activation energy of the initial step of nucleophilic attack, namely deprotonation of terminal DNA primer O3'H group. Thus, subtle differences in the enzyme active-site between matched and mismatched base pairs generate significant differences in catalytic performance.

Binding Sites↗
PubMed2006

Arenavirus diversity and evolution: quasispecies in vivo.

Arenaviruses exist as viral quasispecies due to the high mutation rates of the low-fidelity viral RNA-dependent RNA polymerase (RdRp). This genomic heterogeneity is advantageous to the population, allowing for adaptation to rapidly changing environments that present varying types and degrees of selective pressure. The significant variation in biological properties observed among lymphocytic choriomeningitis virus (LCMV) strains, the prototypic arenavirus, indicates to what extent a quasis-pecies dynamics may play a role in arenavirus adaptability and pathogenesis. Several aspects of arenavirus variability and its contribution to pathogenesis will be discussed.

Animals↗
PubMed1997

Hepatitis C virus genotypes in Korea and their relationship to clinical outcome in type C chronic liver diseases.

OBJECTIVES: The relationship between HCV genotype and the development of more serious liver disease has not been clearly established. This study was to investigate the distribution pattern of HCV genotypes in Korea and their relationship to the viremic level and to progression of chronic liver disease. METHODS: Study population was 217 patients with type C chronic liver disease. They were divided into 4 groups; 83 patients with near-normal ALT (group 1), 64 patients with elevated ALT (group 2), 20 patients with decompensated liver cirrhosis (group 3) and 50 patients with hepatocellular carcinoma (group 4). HCV genotypes were determined by reverse transcription polymerase chain reaction (RT-PCR) using mixed primer sets, and then the fidelity of genotyping was confirmed by cloning and sequencing. HCV RNA concentration was measured by quantitative competitive RT-PCR for 23 patients in group 2. RESULTS: The genotypes could be determined in 166 (76%) out of 217 patients. Type 1b and type 2a were predominantly occurring over the other types in somewhat similar frequency (45% and 51%, respectively). The genotype distribution of type 1b and 2a among four different groups showed 42% and 54% in group 1, 49% and 45% in group 2, 53% and 47% in group 3 and 41% and 57% in group 4; thus there was no significant difference in genotype distribution among 4 different disease groups. However, the viremia levels in patients with genotype 1b infection were significantly higher than those with genotype 2a. CONCLUSION: Genotype 2a infection is as prevalent as genotype 1b in Korea, and genotype 2a infection may pose no less risk for progression of disease despite lower replication level than genotype lb infection.

Adolescent↗
PubMed1986

Frameshift mutagenesis by eucaryotic DNA polymerases in vitro.

The frequency and specificity of frameshift errors produced during a single round of in vitro DNA synthesis by DNA polymerases-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) have been determined. DNA polymerase-beta is the least accurate enzyme, producing frameshift errors at an average frequency of one error for each 1,000-3,000 nucleotides polymerized, a frequency similar to its average base substitution accuracy. DNA polymerase-alpha is approximately 10-fold more accurate, producing frameshifts at an average frequency of one error for every 10,000-30,000 nucleotides polymerized, a frequency which is about 2- to 6-fold lower than the average pol-alpha base substitution accuracy. DNA polymerase-gamma is highly accurate, producing on the average less than one frameshift error for every 200,000-400,000 nucleotides polymerized. This represents a more than 10-fold higher fidelity than for base substitutions. Among the collection of sequenced frameshifts produced by DNA polymerases-alpha and beta, both common features and distinct specificities are apparent. These specificities suggest a major role for eucaryotic DNA polymerases in modulating frameshift fidelity. Possible mechanisms for production of frameshifts are discussed in relation to the observed biases. One of these models has been experimentally supported using site-directed mutagenesis to change the primary DNA sequence of the template. Alteration of a pol-beta frameshift hotspot sequence TTTT to CTCT reduced the frequency of pol-beta-dependent minus-one-base errors at this site by more than 30-fold, suggesting that more than 97% of the errors at the TTTT run involve a slippage mechanism.

Animals↗
PubMed1981

Evolution and longevity-assurance processes.

The role of the development of information fidelity systems in evolution is explored, with evidence that the loss in the ability to maintain the integrity of homeostasis in organisms over time, aging, is correlated with the loss in the ability to maintain integrity at the molecular biological level. Evolutionary-comparative analysis places an upper limit on the number of these systems important to the evolution of longevity in the primates, and suggests the importance of insuring the stability of information bearing macromolecules in evolution and the role of modulators of damage to these moieties in the expression of the senescent state.

Aging↗
PubMed2006

Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase.

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 microM(-1) s(-1), a value approximately 10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 microM(-1) s(-1), a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli.

Acrolein, a reactive alpha,beta-unsaturated aldehyde found ubiquitously in the environment and formed endogenously in mammalian cells, reacts with DNA to form an exocyclic DNA adduct, 3H-8-hydroxy-3-(beta-D-2'-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (gamma-OH-PdG). The cellular processing and mutagenic potential of gamma-OH-PdG have been examined, using a site-specific approach in which a single adduct is embedded in double-strand plasmid DNA. Analysis of progeny plasmid reveals that this adduct is excised by nucleotide excision repair. The apparent level of inhibition of DNA synthesis is approximately 70% in Escherichia coli DeltarecA, uvrA. The block to DNA synthesis can be overcome partially by recA-dependent recombination repair. Targeted G --> T transversions were observed at a frequency of 7 x 10(-4)/translesion synthesis. Inactivation of polB, dinB, and umuD,C genes coding for "SOS" DNA polymerases did not affect significantly the efficiency or fidelity of translesion synthesis. In vitro primer extension experiments revealed that the Klenow fragment of polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite gamma-OH-PdG. We conclude from this study that DNA polymerase III catalyzes translesion synthesis across gamma-OH-PdG in an error-free manner. Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine to protect E. coli from the potential genotoxicity of this DNA adduct.

Acrolein↗
PubMed2003

Encapsidation and transduction of cellular genes by retroviruses.

Retroviruses normally package their genomic RNA with high fidelity. However, the fidelity is apparently imperfect, since some cellular mRNA is present in standard retrovirus particles. Further, transcripts originating in the 5' LTR of the integrated provirus sometimes extend beyond the 3' end of the provirus, resulting in the production of chimeric RNAs containing both viral and cellular sequences. These RNAs can be exported to the cytoplasm and packaged into assembling virus particles. When such particles infect a new host cell, reverse transcriptase may copy the cellular sequences, as well as viral sequences, into DNA. In turn, recombinational events during reverse transcription can result in the incorporation of cellular sequences into retroviral genomes. If the cellular sequences encode proteins involved in the control of cell growth, then the high or inappropriate expression of these sequences as part of the retroviral genome may cause the malignant transformation of the infected cell. Viruses of this type, that transduce cellular transforming genes, are known as acute transforming viruses. They can only arise in animals infected with replication-competent retroviruses, and in general cannot produce progeny viruses without replication-competent "helper" viruses. Since they are produced by a complex, multi-step pathway, acute transforming viruses are only generated at very low frequencies.

Animals↗
PubMed1996

Proliferating cell nuclear antigen promotes misincorporation catalyzed by calf thymus DNA polymerase delta.

A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase delta (pol delta) and synthetic oligonucleotide template-primers containing a mispaired nucleotide at the 3'-terminal position of the primer. This complex is indistinguishable in composition from that formed with a fully base paired template-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol delta on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol delta misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol delta, the ability to incorporate (and/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(alpha-phosphonomethyl)-beta, gamma-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol delta.template-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol delta. This model suggests an explicit mechanistic requirement for the intrinsic 3'-5'-exonuclease of pol delta.

Animals↗
PubMed2003

Time versus size: which characteristic of a neural response carries more information?

Considering a variety of quite different candidate neural codes, Cheng and Wasserman [(1996) Biol Cybern 75, 93-103, 105-115] reported data which suggested that amplitude codes transmitted information more faithfully than temporal codes. Reanalyzing their data, the present study measured how well size and time represented information resident within the same neural response feature, namely the response peak. Responses from photoreceptor cells and optic nerve fibers in the peripheral visual system of Limulus polyphemus were therefore re-examined using signal detectability values provided by receiver-operating-characteristic analyses to compare how well the timing and the amplitude of the response peak represent information within a single cell and how well they transmit information between cells. Data were available for several cells in several different light-adaptation states onto which several different test-flash intensities had been superimposed. The present reanalysis of these data replicated the results yielded earlier by the peak candidate code, and compared them with the data produced by two different measurements of the timing of the same response peak. A relative-timing code was derived from measurements of the time that elapsed between the moment when the response exceeded a criterion potential and when it reached its peak. An absolute-timing code was derived from the time that elapsed between test flash onset and the peak. The results clearly indicated that the peak code represented information within cells better than either of the two timing codes. However, both peak and absolute-timing codes clearly transmitted the available information between cells more faithfully than the relative-timing code. These data lead to two conclusions. First, that the same response feature, when measured in different ways, can produce remarkably different information processing outcomes. In this particular case, time represented information resident within a cell less well than did size. Second, that the fidelity of information transmission between nerve cells may be relatively independent of the quantity of information resident within any particular cell.

Models, Neurological↗
PubMed1986

Influence of RNA polymerase II upon vaccinia virus-related translation examined by means of alpha-amanitin.

Our previous studies employing alpha-amanitin-sensitive H-9 and resistant Ama 102 mutant host cells demonstrated that polymerase II (Pol II), or a drug-sensitive component of the enzyme, is required for replication of vaccinia virus. Evidence was also obtained indicating that transcription from the host genome does not appear to be involved (Silver et al., 1979; Silver and Dales, 1982), suggesting a possible role for Pol II in transcription from the viral genome. This idea is consistent with the present findings, based on immunofluorescence analysis, which revealed that upon infection Pol II antigen is mobilized out of the nucleus into discrete cytoplasmic foci. Effects of treating H-9 rat myoblasts with alpha-amanitin upon vaccinia-specific protein synthesis were also examined. Under the experimental conditions employed, the toxin drastically curtailed in vivo translation into early, late and late-late proteins without altering the spectrum of polypeptides produced. By contrast, treatment with the drug affected, only minimally, the rate of transcription into viral RNA, whether in vivo or from isolated vaccinia factories. The mRNA isolated from infected and treated or untreated cells was translated in a reticulocyte lysate with equal efficiency and general fidelity. This finding suggests that Pol II may be involved in transcription into RNAs related to factors controlling the in vivo translation process. The possible mechanisms for exercising such controls are discussed in relation to factors regulating transcription by host RNA polymerases from a viral DNA genome.

Amanitins↗
PubMed2002

Selective delivery of nitric oxide to a cellular target: a pseudosubstrate-coupled dinitrosyl-iron complex inhibits the enteroviral protease 2A.

Nitric oxide (NO) regulates multiple biological processes. To use NO as a potential therapeutic substance, a more selective modulation of individual NO targets is desirable. Here, we tested whether peptide conjugation of the dinitrosyl-iron complex (DNIC), a potent NO donor, confers targeted NO delivery. As target, we used the protease 2A of Coxsackie-B-viruses (2A(pro)), which can cause dilated cardiomyopathy. Through S-nitrosylation, NO inhibits this protease, which is essential for viral replication. The tetrapeptide Leu-Ser-Thr-Cys (LSTC) (based on the 2A(pro) substrate recognition motif) and DNIC generated LSTC-DNIC in vitro by S-nitrosylation as evidenced by reverse-phase chromatography. In vitro, LSTC-DNIC (IC(50) 510 nM) dose-dependently inhibited purified 2A(pro) 4.7-fold more effectively than DNIC (IC(50) 2.4 microM), whereas LSTC alone had no effect. In intact cells, expression of Coxsackievirus protease 2A by transient transfection led to eIF4G-I-cleavage. LSTC-DNIC (IC(50) 23 microM) dose-dependently inhibited eIF4G cleavage in 2A(pro)-transfected cells 3.8-fold more effectively than DNIC (IC(50) 88 microM). To test the specificity of the DNIC-conjugated LSTC peptide part, we investigated its influence on Caspase-3, a known target for S-nitrosylation. LSTC-DNIC and DNIC inhibited purified Caspase-3 in vitro (IC(50) 3.7 microM) and in intact cells similarly. LSTC conjugation of DNIC enhances its fidelity for inhibition of 2A(pro) in vitro and intracellularly. Peptide-DNIC may be useful to selectively modulate cellular processes by NO, i.e., to enhance its antiviral properties.

Cysteine Endopeptidases↗
PubMed1988

The fidelity of base selection by the polymerase subunit of DNA polymerase III holoenzyme.

In common with other DNA polymerases, DNA polymerase III holoenzyme of E. coli selects the biologically correct base pair with remarkable accuracy. DNA polymerase III is particularly useful for mechanistic studies because the polymerase and editing activities reside on separate subunits. To investigate the biochemical mechanism for base insertion fidelity, we have used a gel electrophoresis assay to measure kinetic parameters for the incorporation of correct and incorrect nucleotides by the polymerase (alpha) subunit of DNA polymerase III. As judged by this assay, base selection contributes a factor of roughly 10(4)-10(5) to the overall fidelity of genome duplication. The accuracy of base selection is determined mainly by the differential KM of the enzyme for correct vs. incorrect deoxynucleoside triphosphate. The misinsertion of G opposite template A is relatively efficient, comparable to that found for G opposite T. Based on a variety of other work, the G:A pair may require a special correction mechanism, possibly because of a syn-anti pairing approximating Watson-Crick geometry. We suggest that precise recognition of the equivalent geometry of the Watson-Crick base pairs may be the most critical feature for base selection.

DNA Polymerase III↗
PubMed1978

Mechanism of ultraviolet-induced mutagenesis: extent and fidelity of in vitro DNA synthesis on irradiated templates.

The effect of UV irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded varphiX174 phage DNA as substrates. Unfractionated and fractionated cell-free extracts from Escherichia coli pol(+) and polA1 mutants as well as purified DNA polymerase I were used as sources of enzymatic activity. (DNA polymerases, as used here, refer to deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7.) The extent of inhibition of DNA synthesis on UV-irradiated varphiX174 DNA suggested that pyrimidine dimers act as an absolute block for chain elongation by DNA polymerases I and III. Experiments with an irradiated poly(dC) template failed to detect incorporation of noncomplementary bases due to pyrimidine dimers. A large increase in the turnover of nucleoside triphosphates to free monophosphates during synthesis by DNA polymerase I on irradiated varphiX174 DNA has been observed. We propose that this nucleotide turnover is due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite UV photolesions, by the 3'-->5' "proofreading" exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result. In support of this hypothesis, DNA synthesis by DNA polymerase from avian myeloblastosis virus and by mammalian DNA polymerase alpha, both of which are devoid of any exonuclease activity, was found to be only partially inhibited, but not blocked, by UV irradiation of the template and accompanied by an increased incorporation of noncomplementary nucleotides. It is suggested that UV mutagenesis in bacteria requires an induced modification of the cellular DNA replication machinery, possibly an inhibition of the 3'-->5' exonuclease activity associated with DNA polymerases.

Coliphages↗
PubMed2006

Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase.

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu(89) side chain. We investigated the role of Glu(89) in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu(89) can influence the slippage of both strands. Glu(89) substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu(89) on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu(89) and Lys(154), which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.

Base Sequence↗
PubMed1993

Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis.

Conserved site-directed mutations were introduced into the second most conserved amino acid region, region II, of the human DNA polymerase alpha catalytic subunit. These mutants were expressed in the baculovirus system and purified to near homogeneity. The mutants had polymerase activity ranging from 4 to 60% compared with the wild type polymerase alpha. Steady-state kinetic analysis of mutants G841A, D860A, D860S, D860N, Y865S, and Y865F demonstrated no significant difference in their Km values for primer-template compared with that of the wild type enzyme. In contrast, mutants D860A, Y865S, and Y865F showed a 5-10-fold increase in the Km for deoxynucleotide triphosphate (dNTP) compared with the wild type enzyme. DNA synthetic fidelity studies of these mutants showed that mutant Y865S but not Y865F had a greater than 10-fold higher misinsertion efficiency than the wild type enzyme in Mg(2+)-catalyzed reactions. However, with Mn2+ as the metal activator, Y865S and Y865F demonstrated a 2- and 9-fold higher misinsertion efficiency, respectively. These results indicate that Asp860 and Tyr865 in region II of human DNA polymerase alpha are involved in incoming dNTP substrate binding. Using three deoxynucleotide structural analogs as probes, we show that the nucleotide base is the structural requirement for dNTP binding with Tyr865. Furthermore, abolishing the hydrophobic phenyl ring side chain of Tyr865 by replacing tyrosine with serine rendered the enzyme resistant to aphidicolin. Results of these studies strongly suggest that the phenyl ring of Tyr865 directly interacts with the nucleotide base moiety of the dNTP and plays a critical role in the misinsertion fidelity of DNA synthesis. Although mutation of Gly841 to Ala did not affect the binding of primer-template, it had a significant decrease in kcat, an increase in Km for dNTP, a striking decrease of processivity, and also resistance to aphidicolin. Thus, mutation of this residue, Gly841, which is highly conserved among the alpha-like DNA polymerases, appears to affect both catalysis and substrate deoxynucleotide binding. This suggests that Gly841 is essential for the maintenance of the overall structure of the polymerase alpha catalytic site.

Amino Acid Sequence↗