Variety Of DNA Polymerases Research Articles

Genome-wide model for the normal eukaryotic DNA replication fork

To investigate DNA replication enzymology across the nuclear genome of budding yeast, deep sequencing was used to establish the pattern of uncorrected replication errors generated by an asymmetric mutator variant of DNA polymerase δ (Pol δ). Sequencing of 16 genomes identified 1,206-bp substitutions generated over 33 generations by L612M Pol δ in a mismatch repair defective strain. Alignment of sequences flanking these substitutions identified "hotspot" motifs for Pol δ replication errors. The substitutions were distributed evenly across all 16 chromosomes. The vast majority were transitions that occurred with a strand bias that varied in a predictable manner relative to known functional origins of replication. This strand bias strongly supports the idea that Pol δ is primarily a lagging strand polymerase during replication across the entire nuclear genome.

Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs

Bacteriophage ϕ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of ϕ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)(2)] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique ϕ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make ϕ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.

A DNA Polymerase with Increased Reactivity for Ribonucleotides and C5‐Modified Deoxyribonucleotides

Just do it: Directed evolution enables efficient enzymatic synthesis of catalytically active RNA as well as highly functionalized C5-modified DNA. The DNA polymerase variant that is proficient in incorporating nucleobase and sugar-modified nucleotides was identified by a chip-based protein library screening assay. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase

The only Y-family DNA polymerase conserved among all domains of life, DinB and its mammalian ortholog pol kappa, catalyzes proficient bypass of damaged DNA in translesion synthesis (TLS). Y-family DNA polymerases, including DinB, have been implicated in diverse biological phenomena ranging from adaptive mutagenesis in bacteria to several human cancers. Complete TLS requires dNTP insertion opposite a replication blocking lesion and subsequent extension with several dNTP additions. Here we report remarkably proficient TLS extension by DinB from Escherichia coli. We also describe a TLS DNA polymerase variant generated by mutation of an evolutionarily conserved tyrosine (Y79). This mutant DinB protein is capable of catalyzing dNTP insertion opposite a replication-blocking lesion, but cannot complete TLS, stalling three nucleotides after an N(2)-dG adduct. Strikingly, expression of this variant transforms a bacteriostatic DNA damaging agent into a bactericidal drug, resulting in profound toxicity even in a dinB(+) background. We find that this phenomenon is not exclusively due to a futile cycle of abortive TLS followed by exonucleolytic reversal. Rather, gene products with roles in cell death and metal homeostasis modulate the toxicity of DinB(Y79L) expression. Together, these results indicate that DinB is specialized to perform remarkably proficient insertion and extension on damaged DNA, and also expose unexpected connections between TLS and cell fate.

Correction: Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ

Correction: Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ

Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ

BackgroundDNA polymerase lambda (Polλ) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB).Principal FindingsHere, we described a novel natural allelic variant of human Polλ (hPolλ) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Polλ variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPolλ variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPolλ.ConclusionsThe expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.

Efficiency and specificity of microRNA-primed nucleotide analog incorporation by various DNA polymerases

MicroRNAs (miRNAs) as endogenous regulators of gene expression have spurred a surge of interest for their quantification and expression analysis. High-sensitivity and high-specificity miRNA detection techniques, such as real-time polymerase chain reaction and recently introduced bioluminescent miRNA detection, require systematic study of DNA polymerases for use with miRNAs. In this study, a variety of DNA polymerases were studied to assess their capabilities of using miRNA as a primer and incorporating 2′-deoxyadenosine-5′- O-(1-thiotriphosphate) as a dATP alternative during DNA strand extension. Five DNA polymerases were investigated: mesophilic DNA polymerase I large (Klenow) fragment, 3′ → 5′ exo − Klenow DNA polymerase, thermophilic Bst DNA polymerase large fragment, Therminator DNA polymerase, and Taq DNA polymerase. The experimental results show that, except for Taq DNA polymerase, the polymerases can use miRNA as a primer and have both common and divergent properties of the nucleotide analog incorporation and miRNA discrimination. DNA polymerase I large (Klenow) fragment showed no detectable polymerization product with the thio-modified dATP as a substrate. Thermophilic Bst DNA polymerase had the highest specificity for miRNA recognition on a DNA template. The study provides a novel method for miRNA detection without reverse transcription to complementary DNA that is faster, simpler, and less prone to biases and errors.

The I260Q Variant of DNA Polymerase β Extends Mispaired Primer Termini Due to Its Increased Affinity for Deoxynucleotide Triphosphate Substrates

DNA polymerase beta plays a key role in base excision repair. We have previously shown that the hydrophobic hinge region of polymerase beta, which is distant from its active site, plays a critical role in the fidelity of DNA synthesis by this enzyme. The I260Q hinge variant of polymerase beta misincorporates nucleotides with a significantly higher catalytic efficiency than the wild-type enzyme. In the study described here, we show that I260Q extends mispaired primer termini. The kinetic basis for extension of mispairs is defective discrimination by I260Q at the level of ground-state binding of the dNTP substrate. Our results suggest that the hydrophobic hinge region influences the geometry of the dNTP binding pocket exclusively. Because the DNA forms part of the binding pocket, our data are also consistent with the interpretation that the mispaired primer terminus affects the geometry of the dNTP binding pocket such that the I260Q variant has a higher affinity for the incoming dNTP than wild-type polymerase beta.

Towards the Replication of xDNA, a Size-expanded Unnatural Genetic System

Here we study the viability of an unnatural genetic system with size-expanded geometry (xDNA). xDNA contains base pairs 2.4 A larger than those of natural DNA. The expanded geometry is expected to be problematic for the natural high-fidelity replication machinery required to process genetic information. However, initial studies with a variety of DNA polymerases are promising in demonstrating replication of these unnatural bases. The results suggest the future possible viability of fully functional unnatural genetic systems, and give insight into the steric limits of some natural DNA polymerases.

Streptococcus agalactiae DNA polymerase I is an efficient reverse transcriptase

Genome annotations result generally from large sequence alignments by bioinformatics. Large scale biochemical data are more difficult to obtain. They derive for example from directed protein evolution experiments by selection. A previously reported directed enzyme evolution experiment by in vitro selection of Stoffel fragment variants of Taq DNA polymerase I was used here to predict the activities of Streptococcus agalactiae DNA polymerase I. The reverse transcriptase activity of S. agalactiae DNA polymerase I was measured and the kinetic parameters for this RNA-dependent DNA polymerase are given. RNA-templated DNA repair is suggested as a possible biological function for this biochemical activity.

Micrmolluscs in molecular systematics: Experiences and best practices

The use of molecular techniques is pervasive in contemporary systematics. We stress the importance of depositing voucher material, particularly for the less well-known micromolluscs, along with images from which specimens can be identifi ed. Appropriate storage is paramount to ensure long-term survival of those voucher specimens. We detail methods of animal extraction that retain the shell as a voucher specimen, and detail salvage of radula and operculum from DNA spin columns. DNA can be extracted from freshly collected micromolluscs, but most dry and wet material in museum collections is not usable for molecular studies. Fixation problems may arise due to bulk collection of material in formalin, washing marine samples in fresh water (osmotic shock), and large surface to volume ratios of micromollusc animals and possible oxidative processes. The most successful strategy to PCR-amplify diffi cult fragments is the use of multiple thermostable DNA polymerase variants.

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities.

Prostate-Cancer-Associated I260M Variant of DNA Polymerase β Is a Sequence-Specific Mutator

Studies show that 30% of 189 tumors sequenced to date express variants of the polymerase beta (pol beta) protein that are not present in normal tissue. This raises the possibility that variants of pol beta might be linked to the etiology of cancer. Here, we characterize the I260M prostate-cancer-associated variant of pol beta. Ile260 is a key residue of the hydrophobic hinge that is important for the closing of the polymerase. In this study, we demonstrate that the I260M variant is a sequence context-dependent mutator polymerase. Specifically, I260M is a mutator for misalignment-mediated errors in dipyrimidine sequences. I260M is also a low-fidelity polymerase with regard to the induction of transversions within specific sequence contexts. Our results suggest that the hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis. Importantly, characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer.

Structure of the 1,4-bis(2'-deoxyadenosin-N6-yl)-2R,3R-butanediol cross-link arising from alkylation of the human N-ras codon 61 by butadiene diepoxide.

The solution structure of the 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2R,3R-butanediol cross-link arising from N(6)-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO(2)) was determined in the oligodeoxynucleotide 5'-d(CGGACXYGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained codon 61 (underlined) of the human N-ras protooncogene. The cross-link was accommodated in the major groove of duplex DNA. At the 5'-side of the cross-link there was a break in Watson-Crick base pairing at base pair X(6).T(17), whereas at the 3'-side of the cross-link at base pair Y(7).T(16), base pairing was intact. Molecular dynamics calculations carried out using a simulated annealing protocol, and restrained by a combination of 338 interproton distance restraints obtained from (1)H NOESY data and 151 torsion angle restraints obtained from (1)H and (31)P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X(6).T(17), accompanied by a shift in the phosphodiester backbone torsion angle beta P5'-O5'-C5'-C4' at nucleotide X(6). The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N(6),N(6)-dA cross-links, which did not predict DNA bending. The rMD calculations suggested the presence of hydrogen bonding between the hydroxyl group located on the beta-carbon of the four-carbon cross-link and T(17) O(4), which perhaps stabilized the base pair opening at X(6).T(17) and protected the T(17) imino proton from solvent exchange. The opening of base pair X(6).T(17) altered base stacking patterns at the cross-link site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this cross-link is bypassed by a variety of DNA polymerases, yet is significantly mutagenic [Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580].

A Method for Detecting Complex Vertebral Malformation in Holstein Calves using Polymerase Chain Reaction–Primer Introduced Restriction Analysis

Complex vertebral malformation (CVM), a hereditary lethal disease in Holstein calves, is characterized by complex anomalies of the vertebral column and limbs in an aborted fetus and in prematurely born, stillborn, and neonatal calves. The mode of inheritance of CVM is autosomal recessive, and CVM is caused by a point mutation from G to T at nucleotide position 559 of the bovine solute carrier family 35 member 3 (SLC35A3) gene. Although an allele-specific polymerase chain reaction (AS-PCR) is a useful method for diagnosis of CVM, the AS-PCR requires selected DNA polymerases and strictly controlled reaction conditions to obtain reliable results. Therefore, an alternative screening method for the CVM gene would be useful. Polymerase chain reaction-primer introduced restriction analysis (PCR-PIRA) is a method that can be used for detecting a single nucleotide mutation in any gene without a restriction site around the mutation site. In this study, primers were designed to introduce PstI or EcoT22 sites into PCR products from the wild-type and CVM alleles, respectively. The wild-type allele, a heterozygote, and a homozygote of the CVM allele could be discriminated by restriction fragment length polymorphism analysis. Specific introduction of restriction sites into PCR products depending on the change in a single nucleotide of template was shown using a variety of DNA polymerases and PCR machines. Therefore, the PCR-PIRA technique using primers designed in this study might provide a more useful method for extensive screening of CVM.

Expression and purification of histidine-tagged bacteriophage T7 DNA polymerase

The formation of inclusion bodies is a frequent consequence of high-level production of foreign protein in the cytoplasm of Escherichia coli. This phenomenon is also observed with bacteriophage T7 gene 5 protein, the phage-encoded subunit of T7 DNA polymerase, if expression is based on the T5 promoter/ lac operator transcription–translation system present in a vector with ColE1 origin of replication. To avoid tedious procedures for recovering protein from insoluble aggregates, we studied the expression of T7 gene 5 protein using a series of E. coli strains, and optimized the yield of soluble, histidine-tagged (His-tagged) protein by varying the respective growth conditions (temperature, amount of inducer isopropyl-β- d-thiogalactopyranoside, and presence of organic osmolytes). Although the expression levels in three different strains (BL21, SG13009, and XL1-Blue) were almost comparable with a given set of growth conditions, the yields of soluble protein differed markedly. The largest quantities of soluble, His-tagged T7 gene 5 protein were achieved using “cloning strain” XL1-Blue which benefitted significantly from the presence of sorbitol and glycine betaine—in contrast to the expression strains BL21 and SG13009. Purification of His-tagged T7 gene 5 protein was achieved using single-step metal-affinity chromatography that yielded large amounts of highly active polymerase (⩾97% homogeneity). The application of this expression/purification approach represents not only a useful method to purify large quantities of T7 DNA polymerase for structural investigations but also, provides a fast and efficient protocol for the parallel purification of T7 DNA polymerase variants, e.g., for automated screenings or directed evolution experiments.

DNA Polymerase Fidelity: Kinetics, Structure, and Checkpoints

On careful examination of existing kinetic data for correct and incorrect dNTP incorporations by a variety of DNA polymerases, it is apparent that these enzymes resist a unified description. Instead, the picture that emerges is a rather complex one: for most polymerases, there is evidence for a noncovalent step preceding phosphoryl transfer, but there are less reliable data for determining whether the noncovalent step or phosphoryl transfer is rate-limiting during misincorporation. Although the structural conservation in the polymerase superfamily is probably reflected in a common set of intermediates along the reaction pathway, the energetics of these species vary even when closely related polymerases are compared. Consequently, some polymerases apparently show more discrimination between correctly paired and mispaired dNTPs in the binding step, and polymerases may differ in terms of which step of the reaction is rate-limiting in correct and incorrect insertion reactions. Because of the higher energy barrier in the misincorporation reaction, at least some of the intermediates both before and after the rate-limiting step in the misincorporation pathway will have higher energies than the corresponding intermediates in correct incorporation; consequently, these steps can serve as kinetic checkpoints.

A Variant of DNA Polymerase β Is Not Cancer Specific

DNA polymerase β (pol β) carries out base-excision repair (BER) required for DNA maintenance, replication, and recombination in eukaryotic cells. A variant characterized by a deletion of exon 11, an 87-bp region in the catalytic domain (pol β Δ 208–236), was previously described as a possible cause of genomic instability in cancer. The variant form was hypothesized to act in a dominant negative fashion, due to the fact that the variant inhibits the gap filling and DNA binding activities of the wild-type pol β protein. DNA polymerase β transcripts were analyzed in 8 breast cancer cell lines, snap-frozen benign breast tissues from 10 women, and lymphocytes from 10 normal controls, using reverse-transcription polymerase chain reaction (RT-PCR) and three separate primer pairs. The exon 10–12 splice site (variant) was identified using a primer designed to span the spliced exons and by sequencing RT-PCR products that included exon 10, exon 11 (if present), and exon 12. In all of the samples tested, we found both the wild-type and exon 11 87-bp deleted variant mRNAs expressed. We conclude that expression of the DNA polymerase β variant (pol β Δ 208–236) is ubiquitous and not breast cancer specific.

2',5'-linked DNA is a template for polymerase-directed DNA synthesis.

Genomes are composed of nucleic acids bearing 3',5'-phosphodiester-linked sugars. The 2',5'-phosphodiester linkage is a fundamental alternative to the natural linkage and is the predominant product of most nontemplate, nonenzymatic oligomerizations of nucleotide monomers. This fact suggests a chemical bias for the formation of 2',5'-phosphodiester bonds, an important consideration in the context of molecular evolution on Earth or elsewhere. Although 2',5'-linked RNA occurs naturally, it is evidently not used to encode genetic information. Nevertheless, the capacity of the 2',5'-phosphodiester linkage to serve as a genetic material has been probed in laboratory experiments. Initial studies examined both self-pairing and pairing with natural nucleic acids. These studies showed that, while 2',5'-linked DNA and RNA form double and triple helices with themselves, their interactions with natural nucleic acids are asymmetric-pairing is seen with 3',5'-linked RNA but not DNA. Although the ability to form a stable structure (such as a helix) is likely a necessary condition for nonenzymatic information transfer, in the case of enzymatic reactions this precondition may be expected to be lifted in cases where the enzyme can serve as a "template for the template" and make up for structural deficiencies that would be fatal to its abiotic counterpart. In support of this suggestion, we demonstrate that 2',5'-linked DNA is a template for enzymatic synthesis of natural DNA with a variety of DNA polymerases and reverse transcriptases directing the incorporation of all four natural nucleotides with fidelity.

Variants of DNA polymerase Beta extend mispaired DNA due to increased affinity for nucleotide substrate.

DNA polymerase beta offers an attractive system to study the biochemical mechanism of polymerase-dependent mutagenesis. Variants of DNA polymerase beta, Y265F and Y265W, were analyzed for misincorporation efficiency and mispair extension ability, relative to wild-type DNA polymerase beta. Our data show that the fidelity of the mutant polymerases is similar to wild-type enzyme on a one-nucleotide gapped DNA substrate. In contrast, with a six-nucleotide gapped DNA, the mutant proteins are slightly more accurate than the wild-type enzyme. The mutagenic potential of Y265F and Y265W is more pronounced when encountering a mispaired DNA substrate. Here, both variants can extend a G:G mispair quite efficiently, and Y265F can also extend a T:G mispair. The kinetic basis of the increased mispair extension efficiency is due to an improved ability to bind to the incoming nucleotide. Y265W extends the G:G mispair even with an incorrect nucleotide substrate. Overall, our results demonstrate that the Y265 hinge residue is important for stabilizing the architecture of the nucleotide binding pocket of DNA polymerase beta, and that alterations of this residue can have significant impacts upon the fidelity of DNA synthesis.