Rat Pol Beta Research Articles

The Primary DNA-Binding Subsite of the Rat Pol Beta. Energetics of Interactions of the 8-kDa Domain of the Enzyme with the ssDNA

Interactions of the 8-kDa domain of the rat pol β and the intact enzyme with the ssDNA have been studied, using the quantitative fluorescence titration technique. The 8-kDa domain induces large topological changes in the bound DNA structure and engages much larger fragments of the DNA than when embedded in the intact enzyme. The DNA affinity of the domain is predominantly driven by entropy changes, dominated by the water release from the protein. The thermodynamic characteristics dramatically change when the domain is embedded in the intact polymerase, indicating the presence of significant communication between the 8-kDa domain and the catalytic 31-kDa domain. The diminished water release from the 31-kDa domain strongly contributes to its dramatically lower DNA affinity, as compared to the 8-kDa domain. Unlike the 8-kDa domain, the DNA binding of the intact pol β is driven by entropy changes, originating from the structural changes of the formed complexes.

Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase β

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.

Hinge Residue Ile260 of DNA Polymerase β Is Important for Enzyme Activity and Fidelity

DNA polymerases ensure efficient insertion of the correct dNTP into the DNA substrate. They have evolved mechanisms for discriminating among very similar dNTP substrates. DNA polymerase beta is a repair polymerase that provides a model system for a direct study of insertion fidelity. In this study, we examined the role of hinge residue Ile260 of the rat Polbeta on enzyme activity and accuracy. We changed residue I260 to every other amino acid residue and used genetic screens to assess the activity and fidelity of the resulting mutants. The I260D, -E, -K, -N, and -R mutants are significantly less active than wild-type Polbeta. Interestingly, I260H and I260Q are active but exhibit mutator activity. This suggests that the nonpolar nature of residue 260 is important for maintaining the activity and fidelity of Polbeta. We employ molecular modeling as an aid in explaining the observed phenotypes and propose a mechanism whereby the positioning of the DNA substrate in the enzyme and within the surface of the hinge may be a key player in forming an optimal active site for phosphodiester bond formation between Watson-Crick base pairs.

Involvement of an X family DNA polymerase in double-stranded break repair in the radioresistant organism Deinococcus radiodurans.

DNA polymerases of the X family have been implicated in a variety of DNA repair processes in eukaryotes. Here we show that Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced double-stranded DNA breaks, expresses a DNA polymerase belonging to the X family. This novel bacterial polymerase, named PolX(Dr), was identified as the product of the Deinococcal DR0467 gene. The purified PolX(Dr) protein possesses a DNA polymerase activity that is stimulated by MnCl2, a property of the X family DNA polymerases. Antibodies raised against PolX(Dr) recognized human pol lambda, rat pol beta and yeast Pol4 and, conversely, antibodies raised against these proteins recognized PolX(Dr). This immunological cross-reactivity suggests a high degree of structural conservation among the polymerases of the X family. Lack of PolX(Dr) reduced the rate of repair of double-stranded DNA breaks and increased cell sensitivity to gamma-rays. PolX(Dr) thus appears to play an important role in double-stranded DNA break repair in D. radiodurans.

Rat polymerase beta binds double-stranded DNA using exclusively the 8-kDa domain. Stoichiometries, intrinsic affinities, and cooperativities.

Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.

Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

Interactions between rat polymerase beta (pol beta) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol beta complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNA-binding modes of the enzyme, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotides occluded by the protein. The 8-kDa domain of the enzyme engages the double-stranded (ds)DNA downstream from the primer, while the 31-kDa domain has similar affinity for the ss-ds DNA junction and the dsDNA. The affinity of rat pol beta for the gapped DNA is not affected by the size of the gap. The results indicate a plausible model for recognition of the gapped DNA by rat pol beta. The enzyme binds the ss-ds DNA junction of the gap using the 31-kDa domain. This binding induces an allosteric transition, resulting in the association of the 8-kDa domain with the dsDNA, leading to an amplification of the affinity for the gap. The 5' terminal phosphate, downstream from the primer, has little effect on the affinity, but affects the ssDNA conformation of the gap.

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.

The specific inhibitory effect of demethoxydehydroaltenusin, a derivative of dehydroaltenusin, on mammalian DNA polymerase α

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin from the fungus Acremonium sp. was found to be an inhibitor of pol alpha. The present study succeeded in chemically synthesizing dehydroaltenusin, and the compound strongly inhibited calf pol alpha activity and weakly suppressed rat pol beta activity, with IC50 values of 0.68 and 64 microM, respectively. We purified or synthesized various slightly modified derivatives of dehydroaltenusin, and using these, investigated the relationship between chemical structure and the inhibitory effects. These results suggest that the ketone group at the 5'-position in dehydroaltenusin is essential for pol inhibitory activity, and the group at the 5-position is important for the specificity of pol alpha inhibition. Demethoxydehydroaltenusin was found to be the most specific pol alpha inhibitor among the prepared derivatives, and the IC50 values for pols alpha and beta were 0.24 and 89 microM, respectively. This compound did not influence the activities of other replicative pols such as pols delta and epsilon, and also demonstrated no effect on pol alpha activity from another vertebrate, fish and a plant species. Demethoxydehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. Therefore, demethoxydehydroaltenusin is of interest as a mammalian pol alpha-selective inhibitor as a 'chemical knockout agent' in vitro and in vivo.

Increased Activity and Fidelity of DNA Polymerase β on Single-nucleotide Gapped DNA

DNA polymerase beta (pol beta) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol beta activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol beta (kcat/Km,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5'-margin of the gap. pol beta exhibited the highest catalytic efficiency on 5'-phosphorylated 1-nucleotide gapped DNA. This efficiency was >/=500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol beta, as judged by its ability to form G-N mispairs, was also higher (10-100 times) on 5'-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol beta is to fill 5'-phosphorylated 1-nucleotide gaps.

Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity,

Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human pol beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human pol beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol beta is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II beta-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities. A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.

Structure-function analysis of the mammalian DNA polymerase .beta. active site: role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer

The crystal structure of the catalytic domain of rat DNA polymerase beta revealed that Asp256 is located in proximity to the previously identified active site residues Asp190 and Asp192. We have prepared and kinetically characterized the nucleotidyl transfer activity of wild type and several mutant forms of human and rat pol beta. Herein we report steady-state kinetic determinations of KmdTTP, Km(dT)16, and kcat for mutants in residue Asp256 and two neighboring residues, Arg254 and Arg258, all centrally located on strand beta 7 in the pol beta structure. Mutation of Asp256 to alanine abolished the enzymatic activity of pol beta. Conservative replacement with glutamic acid (D256E) led to a 320-fold reduction of kcat compared to wild type. Replacement of Arg254 with an alanine (R254A) resulted in a 50-fold reduction of kcat compared to wild type. The Km(dT)16 of D256E and R254A increased about 18-fold relative to wild type. Replacement of Arg254 with a lysine resulted in a 15-fold decrease in kcat, and a 5-fold increase in the Km(dT)16. These kinetic observations support a role of Asp256 and Arg254 in the positioning of divalent metal ions and substrates in precise geometrical orientation for efficient catalysis. The mutation of Arg258 to alanine (R258A) resulted in a 10-fold increase in KmdTTP and a 65-fold increase in Km(dT)16 but resulted in no change of kcat. These observations are discussed in the context of the three-dimensional structures of the catalytic domain of pol beta and the ternary complex of pol beta, ddCTP, and DNA.

Effects of DNA polymerase beta gene over-expressed in transgenic Drosophila on DNA repair and recombination.

DNA polymerase beta (pol beta) cDNA of rat fused to an enhancer-promoter region plus a poly(A) signal sequence of actin 5C gene of Drosophila (abbreviated pol beta) was transferred to the Drosophila genome. Three of four constructed transgenic strains possessing transgene pol beta on different chromosomes were studied. Levels of the pol beta transcript and those of the polymerization activity of pol beta were markedly elevated in cultured cells transfected with pol beta-bearing vectors as well as in embryos of the transgenic strains. The popular idea that DNA polymerase beta participates in DNA repair was not supported by the observation that a pair of a normal and a pol beta strain, and the other pair of a mei-9 mei-41 (DNA-repair deficient double mutations) strain and a pol beta mei-9 mei-41 strain, showed no difference in survival within each pair after treatment with ultraviolet light, methylmethane sulfonate and mitomycin C. The other idea that DNA polymerase beta participates in recombination was supported by the findings that spontaneous frequency of recombination, either meiotic or mitotic, is significantly higher in a transgenic pol beta strain than in a non-transgenic strain. The enhanced recombination frequency in the pol beta strain may, however, reflect an indirect effect of over-produced pol beta proteins on chromosomal stability. Whatever the direct effect of rat pol beta is, the transgenic pol beta flies will be useful for study of the physiological role of pol beta and the mechanism of recombination.

Rat DNA polymerase β gene can join in excision repair ofEscherichia coli

Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells. To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E. coli defective in a diverse set of enzymatic activities of poll. UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained. Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells.