Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase beta

We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase beta (pol beta) is one of the most important enzymes protecting the cell against DNA damage by base excision repair. In this study, we characterized the inhibitory action of SQMG against rat pol beta. SQMG competed with both the substrate and the template-primer for binding to pol beta. A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol beta, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol beta activity and the inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA. To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping. Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety. We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus of helix-2, and residues in helix-4. Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis. The models show a SQMG interaction interface that is consistent with the data.     

DNA polymerase beta

Mammalian DNA polymerase beta(beta-pol) is a single polypeptide chain enzyme of 39kDa. beta-pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of beta-pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the beta-pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of beta-pol's role in protecting genomic DNA.     

Expression of active rat DNA polymerase beta in Escherichia coli

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.     

Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA

Interactions between the isolated 8-kDa domain of the rat DNA polymerase beta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein-ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m approximately 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.     

Enzymatic properties of rat DNA polymerase beta mutants obtained by randomized mutagenesis

We have used random sequence mutagenesis to generate mutants of DNA polymerase β in an effort to identify amino acid residues important for function, catalytic efficiency and fidelity of replication. A library containing 100 000 mutants at residues 274–278 in the N-helix of the thumb subdomain of the polymerase was constructed and screened for polymerase activity by genetic complementation. The genetic screen identified 4000 active pol β mutants, 146 of which were sequenced. Each of the five positions mutagenized tolerated substitutions, but residues G274 and F278 were only found substituted in combination with mutations at other positions. The least conserved residue, D276, was replaced by a variety of amino acids and, therefore, does not appear to be essential for function. Steady-state kinetic analysis, however, demonstrated that D276 may be important for catalytic efficiency. Mutant D276E exhibited a 25-fold increase in catalytic efficiency over the wild-type enzyme but also a 25-fold increase in G:T misincorporation efficiency. We present a structural model that can account for the observations and we discuss the implications of this study for the question of enzyme optimization by natural selection.     

Structural design of a eukaryotic DNA repair polymerase: DNA polymerase beta

DNA polymerase beta, the smallest eukaryotic DNA polymerase, is designed to synthesize DNA in short DNA gaps during DNA repair. It is composed of two specialized domains that contribute essential enzymatic activities to base excision repair (BER). Its amino-terminal domain possesses a lyase activity necessary to remove the 5'-deoxyribose phosphate (dRP) intermediate generated during BER. Removal of the dRP moiety is often the rate-limiting step during BER. Failure to remove this group may initiate alternate BER pathways. The larger polymerase domain has nucleotidyl transferase activity. This domain has a modular organization with sub-domains that bind duplex DNA, catalytic metals, and the correct nucleoside triphosphate in a template-dependent manner. X-ray crystal structures of DNA polymerase beta, with and without bound substrates, has inferred that domain, sub-domain, and substrate conformational changes occur upon ligand binding. Many of these conformational changes are distinct from those observed in structures of other DNA polymerases. This review will examine the structural aspects of DNA polymerase beta that facilitate its role in BER.     

Arginine methylation regulates DNA polymerase beta

Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.     

RNA aptamers selected against DNA polymerase beta inhibit the polymerase activities of DNA polymerases beta and kappa

DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 10¹² individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polβ active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polβ also inhibited DNA polymerase κ, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polβ and κ, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase actvity in cells.     

Mechanism of nucleotide incorporation in DNA polymerase beta

DNA polymerases play a central role in the mechanisms of DNA replication and repair. Here, we report mechanisms of the beta-polymerase catalyzed phosphoryl transfer reactions corresponding to correct and incorrect nucleotide incorporations in the DNA. Based on energy minimizations, molecular dynamics simulations, and free energy calculations of solvated ternary complexes of pol beta and by employing a mixed quantum mechanics molecular mechanics Hamiltonian, we have uncovered the identities of transient intermediates in the phosphoryl transfer pathways. Our study has revealed that an intriguing Grotthuss hopping mechanism of proton transfer involving water and three conserved aspartate residues in pol beta's active site mediates the phosphoryl transfer in the correct as well as misincorporation of nucleotides. The significance of this catalytic step in serving as a kinetic check point of polymerase fidelity may be unique to DNA polymerase beta, and is discussed in relation to other known mechanisms of DNA polymerases.     

DNA polymerase beta from the rat liver. Isolation, properties and inhibitory analysis of a homogeneous preparation

A simple and reproducible purification procedure of homogeneous DNA polymerase beta from rat liver is developed, including sedimentation and saline extraction of rat liver chromatin, chromatography of the extract on DEAE-cellulose, phosphocellulose, Gel Blue A, and DNA sepharose. The purified enzyme isolated with the 8.4% yield proved to be a homogeneous protein with m.w. 38-40 kDa, specific activity 31 units/g, pI 8.6-8.9. Incorporation of [3H]TTP into activated DNA catalysed by DNA polymerase beta was strongly inhibited by dNTP (3'NH2), ddTTP, dNTP (3'F) and slightly inhibited by aCTP and aNTP (3'NH2).     

DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.     

Involvement of DNA polymerase beta in DNA replication and mutagenic consequences

Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.     

Electrofocusing of hydrolases in rat brush border enterocytes]

Different hydrolases (cellobiase, enterokinase, lactase, leucine aminopeptidase, alcaline phosphatase and trehalase) of the brush border's enterocytes of the rat have been studied by electrofocusing. Every hydrolase is focused in a single peak, the pI of which is given.     

Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.     

Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase beta and DNA-ligase

Synthetic deoxy-oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase beta. High levels of DNA polymerase beta supplementation resulted in gap-filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32-mer). However, at lower concentrations of DNA polymerase beta, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5'-PO4 on the downstream primer and supplementation of aging neuronal extracts with DNA-ligase in addition to recombinant DNA polymerase beta. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase beta and DNA-ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity.