Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5 °C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔH_binding) crosses zero (T_H) and becomes favorable (negative). This T_H/activity upshift temperature is 15 °C for Klenow and 30 °C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG^‡, ΔH^‡, and TΔS^‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp^‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH^‡) switches from positive to negative.     

Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases

DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity.     

Amplification efficiency of thermostable DNA polymerases

The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA polymerase efficiencies were determined under identical conditions using five different amplicon templates that varied in length or percentage GC content. Pfu- and Taq-based formulations showed similar efficiencies when amplifying shorter targets (<900 bp) with 45 to 56% GC content. However, when amplicon length or GC content was increased, Pfu formulations with dUTPase exhibited significantly higher efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss the implications of these results.     

Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract

Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of -[³²P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3 side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.     

Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP

The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a basic patch of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.     

Plastid DNA polymerases from higher plants, Arabidopsis thaliana

Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H(2)O(2). These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.     

Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.     

The excision of AP sites by the 3'-5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I

The 3' AP endonucleases (class I) are said to hydrolyze the phosphodiester bond 3' to AP sites yielding 3'-OH and 5'-phosphate ends; on the other hand, the resulting 3' terminal AP site is not removed by the 3'-5' exonuclease activity of the Klenow fragment [1]. We show that AP sites in DNA are easily removed by the 3'-5' exonuclease activity of the Klenow fragment and that they are excised as deoxyribose-5-phosphate. It is suggested that the 3' AP endonucleases are perhaps not the hydrolases they are supposed to be.     

DNA polymerase V-dependent mutator activity in an SOS-induced Escherichia coli strain with a temperature-sensitive DNA polymerase III.

The temperature-sensitive DNA polymerase III (Pol III) encoded by the dnaE486 allele confers a spontaneous mutator activity in SOS-induced bacteria that is largely dependent upon DNA polymerase V (Pol V), encoded by umuD, C. This mutator activity is influenced by the defective proof-reading sub-unit of Pol III encoded by the dnaQ905 (mutD5) allele arguing that Pol V is most likely fixing mutations arising from mismatched primer termini produced by Pol III(486). The size of the dnaQ effect is, however, modest leaving open the possibility that Pol V may be responsible for some of the mutator effect by engaging in bursts of processive activity.     

DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.     

The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum

Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an ∼ 35-kb circular DNA genome of unusually high A/T content (> 86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3′ → 5′ exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T → C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication.     

Involvement in DNA repair of the ruvA gene of Escherichia coli

Summary The ruv operon of Escherichia coli consists of two genes, orfl1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orfl and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.     

Inducible SOS response system of DNA repair and mutagenesis in Escherichia coli

Chromosomal DNA is exposed to continuous damage and repair. Cells contain a number of proteins and specific DNA repair systems that help maintain its correct structure. The SOS response was the first DNA repair system described in Escherichia coli induced upon treatment of bacteria with DNA damaging agents arrest DNA replication and cell division. Induction of the SOS response involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repair, replication, mutagenesis and metabolism of DNA. Under normal growth conditions the SOS genes are expressed at a basal level, which increases distinctly upon induction of the SOS response. The SOS-response has been found in many bacterial species (e.g., Salmonella typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not in eukaryotic cells. However, species from all kingdoms contain some SOS-like proteins taking part in DNA repair that exhibit amino acid homology and enzymatic activities related to those found in E. coli. but are not organized in an SOS system. This paper presents a brief up-to-date review describing the discovery of the SOS system, the physiology of SOS induction, methods for its determination, and the role of some SOS-induced genes.     

DNA sequence analysis of spontaneous mutation in a PolA1 strain of Escherichia coli indicates sequence-specific effects

Summary The sequences of a collection of 261 spontaneous lacI^- mutants recovered in a PolA^- strain of Escherichia coli have indicated an increase in the frequency of most classes of mutation in this strain. Among base substitutions in lacI, a preference for transversions over transitions was observed. In addition, a single transition in the lac operator was enhanced 8-fold. More significantly, of 18 frameshifts, 12 occurred adjacent to a 5-GTGG-3 sequence. Likewise, 15 of 24 deletions and 2 of 10 duplications had 5-GTGG-3 sequences at one or both endpoints. We speculate that the prevalence of mutations at these specific sequences reflects the persistence of strand discontinuities that enhance the opportunity for mutagenic mishaps. Further, 5-GTGG-3 sequences apparently represent sites where DNA polymerase I is involved in some aspect of DNA metabolism. These results strengthen the view that DNA context contributes an important component to spontaneous mutagenesis and indicate an anti-mutagenic role for DNA polymerase I.     

Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli

The replication period of Escherichia coli cells grown in rich medium lasts longer than one generation. Initiation thus occurs in the 'mother-' or 'grandmother generation'. Sister origins in such cells were found to be colocalized for an entire generation or more, whereas sister origins in slow-growing cells were colocalized for about 0.1-0.2 generations. The role of origin inactivation (sequestration) by the SeqA protein in origin colocalization was studied by comparing sequestration-deficient mutants with wild-type cells. Cells with mutant, non-sequesterable origins showed wild-type colocalization of sister origins. In contrast, cells unable to sequester new origins due to loss of SeqA, showed aberrant localization of origins indicating a lack of organization of new origins. In these cells, aberrant replisome organization was also found. These results suggest that correct organization of sister origins and sister replisomes is dependent on the binding of SeqA protein to newly formed DNA at the replication forks, but independent of origin sequestration. In agreement, in vitro experiments indicate that SeqA is capable of pairing newly replicated DNA molecules.     

Optimization of the masking molecule for active-site-protected immobilization of Taq DNA polymerase and its application

The method of oriented and activity-preserved immobilization of biologically active proteins based on concepts of active-site masking and kinetic control was further developed in this study. Minimal requirements for the masking DNA molecule were found to be a 5′overhang of 5–7 nucleotides and a double-stranded region of 11–13 bp to retain approximately 70% of the enzyme activity. The amplification range of protected immobilized (PIM) Taq DNA polymerase was over 1.2 kb. These data suggest that PIM Taq DNA polymerase can be used for various commercial applications.     

Temperature dependence of accuracy of DNA polymerase I from Geobacillus anatolicus

Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was cloned and purified. The accuracy of GF was measured in vitro at three different temperatures under single turnover conditions as well as using a forward mutation assay. In pre-steady-state kinetic measurements, when temperature was raised from 22 °C to 50 °C, the rate (k_pol) for cognate dTTP and non-cognate dATP nucleotide incorporations increased six- and four-fold, respectively, whereas the K_d for both nucleotide incorporations changed only slightly. As a result, the error frequency was remained constant (∼4 × 10⁻⁴) over this temperature range. The accuracy of GF was also measured using a forward mutation assay during a single cycle of DNA synthesis of the lacZα complementation gene in M13mp2 DNA. In this assay, which scores various types of replication errors, mutant frequency of GF was 5 × 10⁻³ at 72 °C which is four-fold higher than that of 37 °C.     

Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases α and β

Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryotic DNA polymerases α (cytosine arabinoside) and β (dideoxythymidine) blocked different steps of DNA repair, demonstrated by the effects of the inhibitors on the relaxation of supercoiled DNA nucleoids following treatment of human cell cultures with ultraviolet light (1–3 J/m²) or MNNG (5 or 20 μM) and the subsequent restoration of the supercoiled nucleoids during repair incubation. Changes in the supercoiling of nucleoid DNA were assayed by analysis of their sedimentation profiles in 15–30% neutral sucrose gradients. Inhibition of repair by novobiocin was partially reversible; upon its removal from the culture medium, the nucleoid DNA of repairing cells became relaxed. The DNA polymerase inhibitors allowed the initial relaxation of DNA after treatment of the cells with ultraviolet or MNNG but delayed the regeneration of rapidly-sedimenting (supercoiled) nucleoid DNA for 2–4 h. Dideoxythymidine (1 mM) was more effective than cytosine arabinoside (1 μM) in producing this delay, but neither inhibitor by itself blocked repair permanently. Incubation of ultraviolet-irradiated cells with 1 μM cytosine arabinoside plus 1 mM dideoxythymidine blocked the completion of repair for 24 h, whereas incubation with 10 μM cytosine arabinoside or 5 mM dideoxythymidine produced only temporary repair delays of 2–4 h. Thus, it is likely that the two DNA polymerase inhibitors act upon separate targets and that both targets are involved in repair. It is concluded from these and from previous studies that (1) the DNA repair-sensitive target of novobiocin and nalidixic acid in vivo is not a DNA polymerase, but, rather, a DNA topoisomerase; (2) this target affects an initial step of DNA repair leading to the relaxation of supercoiled DNA; (3) the DNA polymerization step of repair may involve both α- and β-type DNA polymerases; and (4) in repair, one type of DNA polymerase may substitute for another.     

A rapid procedure to purify Escherichia coli DNA topoisomerase I

On the basis of the asymmetrical charge distribution of Escherichia coli DNA topoisomerase I, we developed a new procedure to purify E. coli DNA topoisomerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-Sepharose FF and Q-Sepharose FF columns. The E. coli DNA topoisomerase I purified here is free of DNase contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were also determined.