DNA polymerase mu (Pol micro), homologous to TdT, could act as a DNA mutator in eukaryotic cells
2000; Springer Nature; Volume: 19; Issue: 7 Linguagem: Inglês
10.1093/emboj/19.7.1731
ISSN1460-2075
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle3 April 2000free access DNA polymerase mu (Pol μ), homologous to TdT, could act as a DNA mutator in eukaryotic cells Orlando Domínguez Orlando Domínguez Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author José F. Ruiz José F. Ruiz Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Teresa Laín de Lera Teresa Laín de Lera Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Miguel García-Díaz Miguel García-Díaz Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Manuel A. González Manuel A. González Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Tomas Kirchhoff Tomas Kirchhoff Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Carlos Martínez-A Carlos Martínez-A Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Antonio Bernad Antonio Bernad Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Luis Blanco Corresponding Author Luis Blanco Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Orlando Domínguez Orlando Domínguez Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author José F. Ruiz José F. Ruiz Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Teresa Laín de Lera Teresa Laín de Lera Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Miguel García-Díaz Miguel García-Díaz Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Manuel A. González Manuel A. González Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Tomas Kirchhoff Tomas Kirchhoff Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Carlos Martínez-A Carlos Martínez-A Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Antonio Bernad Antonio Bernad Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Luis Blanco Corresponding Author Luis Blanco Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain Search for more papers by this author Author Information Orlando Domínguez1, José F. Ruiz1, Teresa Laín de Lera2, Miguel García-Díaz1, Manuel A. González2, Tomas Kirchhoff1, Carlos Martínez-A2, Antonio Bernad2 and Luis Blanco 1 1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain 2Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología (CSIC), Universidad Autónoma, 28049 Madrid, Spain ‡O.Domínguez and J.F.Ruiz contributed equally to this work. *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:1731-1742https://doi.org/10.1093/emboj/19.7.1731 This work is dedicated to the memory of Professor Eladio Viñuela PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol μ), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol μ, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn2+ ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol μ was enhanced by the presence of a template strand. Using activating Mg2+ ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn2+ ions, template-enhanced polymerization produced a random insertion of nucleotides. Northern-blotting and in situ analysis showed a preferential expression of Pol μ mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol μ, present in the databases, derived from germinal center B cells. Therefore, Pol μ is a good candidate to be the mutator polymerase responsible for somatic hypermutation of immunoglobulin genes. Introduction The maintenance and stability of genetic information in DNA depends largely on the high fidelity of DNA synthesis displayed by replicative polymerases, which in most cases are capable of proofreading insertion errors (reviewed by Bebenek and Kunkel, 1995). A further improvement in DNA stability relies on different systems of DNA repair, able to detect and repair most kinds of DNA damage that, if left unrepaired, could lead to cell transformation and death. Despite all this DNA maintenance enzymology, capable of excising bases and nucleotides, repairing breaks and correcting mismatches, there is some 'necessary risk' of accumulating DNA mutations as the driving force allowing evolution. Such background mutability could be due to misfunction of replication and the repair machinery, but also to the participation of a specific enzymology, contributed by mutator (error-prone) DNA polymerases, which should work in opposition to DNA repair. DNA polymerase beta (Pol β) is one of the known cellular DNA polymerases for which a specific role in DNA repair has been proposed (Wilson, 1998; Dianov et al., 1999). However, a working hypothesis is that some altered versions of Pol β could represent mutator DNA polymerases acting as dominant error-prone enzymes capable of altering the normal levels of DNA repair (Bhattacharyya and Banerjee, 1997; Clairmont and Sweasy, 1998; Clairmont et al., 1999). Recently, three novel non-essential cellular DNA polymerases, zeta (ζ; Lawrence and Hinkle, 1996), eta (η; Johnson et al., 1999a) and theta (θ; Sharief et al., 1999) have been reported. Pol ζ and Pol η are capable of altering the outcome of the DNA repair process, since these enzymes are able to use damaged (unrepaired) DNA efficiently as a template (see Friedberg and Gerlach, 1999). In yeast and fungi, most spontaneous and damage-induced mutations are introduced by Pol ζ responsible for trans-lesion DNA synthesis (Han et al., 1998). A model for somatic hypermutation of Ig genes has been proposed whereby Pol ζ is recruited to the Ig locus (Diaz and Flajnik, 1998). Pol η is required for error-free replication of UV lesions, and its absence produces the variant (V) form of xeroderma pigmentosum, an autosomal recessive disease characterized by a high incidence of skin cancers (Johnson et al., 1999b; Masutani et al., 1999). More recently, a novel DNA polymerase homologous to Pol β, named Pol lambda (λ), has been described and predicted to be involved in DNA repair synthesis during meiotic recombination (M.García-Díaz, O.Domínguez, L.A.López–Fernández, T.Laín de Lera, M.L.Saníger, J.F.Ruiz, M.Párraga, M.J.García, T.Kirchhoff, J.del Mazo, A.Bernad and L.Blanco, submitted). A clear example of the existence of a particular enzymology for the generation of diversity is the enzyme terminal deoxynucleotidyltransferase (TdT), although its action appears to be restricted to specific genes such as those coding for antigen receptors (reviewed in Bentolila et al., 1995). TdT is a DNA-independent DNA polymerase, i.e. it has the ability to add nucleotides to DNA without any template information. This unusual ability is exploited at the broken ends of the V(D)J recombination intermediates of rearranging antigen receptor genes. The specificity of TdT action on these intermediates depends not only on its expression pattern, restricted to primary lymphoid organs, but also on specific interactions with the Ku protein, the DNA-binding component of DNA-dependent protein kinase (DNA-PK), which functions in DNA repair, V(D)J recombination and isotype switching (Mahajan et al., 1999). Interestingly, TdT and Pol β are evolutionarily related, belonging to the family X of DNA polymerases (see Ito and Braithwaite, 1991). We describe here the identification and preliminary biochemical characterization of a novel human DNA polymerase, named DNA polymerase mu (Pol μ), with a high similarity to TdT, and whose mRNA is highly expressed in secondary lymphoid tissues. The purified enzyme exhibited both terminal deoxynucleotidyltransferase activity and unprecedented error-proneness on primer–template structures. We propose that Pol μ could act as a DNA mutator polymerase responsible for the somatic hypermutation of immunoglobulin genes. Results Identification of a novel terminal transferase in human cells The identification and cloning of the complete cDNA sequence of the novel human DNA polymerase Pol μ belonging to family X was carried out as described in Materials and methods. Pol μ is closely related to TdT, a member of the Pol X family whose template-independent polymerization capacity contributes to generation of diversity in antigen receptor genes (reviewed in Bentolila et al., 1995). Figure 1 shows a multiple alignment of human Pol μ and TdTs from different origins, demonstrating an overall amino acid identity of 41%. This value is significantly higher than that relating Pol μ and Pol β (23%) or Pol β and TdT (22%). A nuclear localization signal (NLS) of the most common type (SV40 large T antigen) is predicted at the sequence 'PKRRRAR' (residues 3–9) of Pol μ. A similar sequence in TdTs was proposed to act as an NLS (Bentolila et al., 1995). Interestingly, residues 22–120 of Pol μ are predicted (see Materials and methods) to form a BRCT domain (Bork et al., 1997; Callebaut and Mornon, 1997). This domain, whose name derives from its initial identification at the C–terminal domain of the BRCA1 protein, is proposed to mediate protein–protein interactions in a variety of proteins involved in DNA repair and cell cycle checkpoint regulation upon DNA damage (Bork et al., 1997). As indicated in Figure 1, Pol μ residues 141–494 form a conserved Pol β core, whose three-dimensional structure in the presence of DNA and ddCTP has been solved at high resolution (Pelletier et al., 1994). Pol μ shares 139 of the 209 amino acid residues (66%) that are invariant among TdTs from very different origins, implying evolutionarily conserved structural and perhaps functional relationships. Pol μ also conserves 21 of the 23 amino acid residues that are conserved among all members of the heterogeneous Pol X family, including all those residues acting as metal, dNTP and DNA ligands, or triggering conformational changes upon ternary complex formation (see legend to Figure 1; for a review, see Oliveros et al., 1997). Therefore, as will be described later, characterization of the polymerization activity associated with Pol μ was necessary in order to determine whether this TdT homologue has terminal deoxynucleotidyltransferase activity. Figure 1.Pol μ, a novel eukaryotic DNA polymerase homologous to TdT. Multiple alignment of human Pol μ (this study) with TdTs from human (Hs; sp P04053), bovine (Bt; sp P06526), murine (Mm; sp P09838), Monodelphis domestica (Md; sp Q02789), chicken (Gd; sp P36195) and Xenopus laevis (Xl; sp P42118). Numbers between slashes indicate the amino acid position relative to the N-terminus of each DNA polymerase. A putative nuclear localization signal (NLS) at residues 3–9 of human Pol μ is boxed. Amino acid residues 22–118 of Pol μ (boxed) are predicted to form a BRCT domain (Bork et al., 1997). Amino acid residues 141–494 of Pol μ (boxed) form a conserved Pol β core (see text for details). Invariant residues between Pol μ and TdTs are indicated with white letters (on a black background). Identical residues among TdTs are in bold and boxed (grey). Other relevant similarities between Pol μ and TdTs are in bold. Conservative substitutions were considered as follows: K, H and R; D, E, Q and N; W, F, Y, I, L, V, M and A; G, S, T, C and P. The 23 residues that are invariant among DNA polymerase X members (Oliveros et al., 1997) are indicated with an asterisk. Dots at the bottom of the alignment indicate putative homologues to Pol β residues (Pelletier et al., 1994) shown to act either as DNA ligands (Gly64, Gly66, Gly105, Gly109, Lys234, Arg254, Arg283 and Tyr296; grey), or as dNTP and metal ligands (Phe272, Gly274, Arg183; Asp190, Asp192 and Asp256; black). Squares at the bottom of the alignment indicate putative homologues to Pol β residues involved in interactions between the 'palm' and 'thumb' subdomains (Gly179/Phe272; Arg182/Glu316). The total length, in number of amino acid residues, is indicated in parentheses. Download figure Download PowerPoint Chromosomal mapping of human Pol μ The human gene (POLM) coding for Pol μ was mapped initially to chromosome 7 by using a panel of human–rodent somatic cell hybrids (see Materials and methods). By radiation hybrid analysis (see Materials and methods), the SHGC marker that best linked with the POLM gene was SHGC–6115, with a lod score of 8.2. Based on the correspondence of this marker with the GCK gene, the POLM gene has been mapped within band 7p13. This region constitutes one of the four known fragile sites in lymphocytes, with a high incidence of molecular alterations such as deletions, inversions and translocations. DNA polymerase activity associated with Pol μ Human Pol μ was overproduced in Escherichia coli and purified to homogeneity as described in Materials and methods. The 55 kDa recombinant protein was obtained in soluble form in a high yield (see Figure 2A). Taking into account that family X DNA polymerases are low processive enzymes with no proofreading 3′–5′ exonuclease, assay conditions were selected to favour detection of a TdT-related enzyme such as Pol μ versus endogenous E.coli DNA polymerases. Thus, labelling of activated (gapped) heteropolymeric DNA was assayed in the presence of a low concentration of dATP as a single nucleotide, and activating Mn2+ ions. These conditions would favour incorporation of complementary and non-complementary nucleotide by terminal deoxynucleotidyltransferases (DNA independent), and by low-fidelity DNA-dependent DNA polymerases without proofreading activity. As shown in Figure 2B, under these conditions, DNA labelling with commercial TdT was ∼10–fold more efficient using Mn2+ rather than Mg2+ activating ions; on the other hand, the opposite metal preference was obtained when labelling with the Klenow fragment of E.coli Pol I. As shown in Figure 2C, DNA labelling activity was detected in the 50% ammonium sulfate precipitate corresponding to the Pol μ-induced extracts, but it was very low in the corresponding uninduced fraction. Interestingly, the catalytic efficiency of the induced DNA polymerase was 20-fold higher in the presence of Mn2+ versus Mg2+ ions. This induced DNA polymerase activity was co-purified with the overproduced 55 kDa polypeptide throughout the purification procedure. As a further demonstration that the induced DNA polymerase activity was intrinsic to Pol μ, the heparin–Sepharose fraction (HS) was sedimented on a glycerol gradient. As shown in Figure 3, a DNA polymerase activity, preferentially activated by Mn2+ ions, co-sedimented perfectly at a molecular weight corresponding to the monomeric form of the Pol μ polypeptide. No 3′–5′ exo- or endonucleolytic activities were associated with the Pol μ peak (data not shown) and, therefore, the glycerol gradient fractions 9 and 10 (pooled) were used as the enzyme source for further activity assays. Figure 2.Expression of human Pol μ in Escherichia coli. (A) Coomassie Blue staining after SDS–PAGE separation of control non-induced (NI) and IPTG-induced (I) extracts of E.coli BL21(DE3) cells transformed with the recombinant plasmid pRSET-hPolμ, and further purification steps of the latter extracts. The mobility of the induced protein Pol μ was compatible with its deduced molecular mass (55 kDa/494 amino acids). After PEI precipitation of the DNA, Pol μ was precipitated with 50% ammonium sulfate (AS), and purified further by phosphocellulose (PC) and heparin–Sepharose (HS) chromatography, as described in Materials and methods. The electrophoretic migration of a collection of molecular mass markers (MW) is shown at the left. (B) Relative activation by Mg2+ versus Mn2+ of TdT and Klenow enzymes during DNA polymerization ([α-32P]dATP labelling) on activated DNA. TdT (5 U) and Klenow (1 U) were assayed for 30 min at 37°C, in the presence of either 10 mM MgCl2 or 1 mM MnCl2 as a source of activating metal ions. DNA polymerase activity, expressed as dAMP incorporation, was quantitated as described in Materials and methods. (C) DNA polymerization activity associated with Pol μ expression. The 50% AS fraction corresponding to either non-induced (N.I.) or induced extracts was assayed and quantitated as described in (B). Download figure Download PowerPoint Figure 3.Co-sedimentation of a DNA polymerase activity with the Pol μ polypeptide. The heparin–Sepharose fraction (HS) shown in Figure 2A was sedimented on a glycerol gradient (15–30%) and fractionated as described in Materials and methods. The inset shows an SDS–PAGE analysis followed by Coomassie Blue staining of some selected fractions. Fractions are numbered from the bottom (1) to the top (22). Arrows indicate the sedimentation position of several molecular mass markers centrifuged under identical conditions. Quantitation of the Pol μ band corresponding to each fraction is expressed in arbitrary units of optical density (a.u.; right ordinates). DNA polymerase activity ([α-32P]dATP labelling of activated DNA) of each fraction, assayed for 15 min at 37°C in the presence of 1 mM MnCl2 (see Materials and methods), is expressed as dAMP incorporation (left ordinates). Download figure Download PowerPoint Pol μ displays terminal deoxynucleotidyltransferase activity but requires a template–primer structure for optimal activity In agreement with the structural similarity of Pol μ and TdT, a terminal deoxynucleotidyltransferase activity associated with Pol μ was demonstrated by using different oligonucleotides as single-stranded primer substrates, again in the presence of Mn2+ as the preferred cation. As shown in Figure 4A, Pol μ was able to catalyse polymerization of any of the four dNTPs to a single-stranded DNA primer in the absence of a template. The catalytic efficiency of the terminal deoxynucleotidyltransferase activity of Pol μ varied as a function of the nucleotide used, dTTP and dCTP (both pyrimidines) being inserted the most efficiently, and dATP the least efficiently. A different nucleotide preference was observed when using TdT, dGTP and dCTP being the preferred nucleotide substrates under these conditions. The terminal deoxynucleotidyltransferase associated with Pol μ was also active, although less efficient, on double-stranded DNA substrates where the primer terminus was paired with a 5′-terminal complementary nucleotide (blunt-ended), a behaviour already described for TdT (results not shown). Figure 4.Pol μ has terminal transferase activity, but requires a template–primer structure for optimal efficiency. (A) Terminal transferase activity associated with human Pol μ. The assay was carried out as described in Materials and methods, using 3.2 nM 5′-labelled single-stranded 19mer (P19) as substrate, 1 mM MnCl2 as a source of activating metal ions, 80 μM each individual deoxynucleotide, and either TdT (2.5 U/41 ng) or Pol μ (20 ng). A control reaction in the absence of enzyme (C) was also carried out. After incubation for 30 min at 30°C, extension of the 5′-labelled oligonucleotide was analysed by 8 M urea–20% PAGE and autoradiography. (B) Template-dependent polymerization catalysed by Pol μ. Polymerization efficiency was assayed comparatively on either poly(dA) (○), oligo(dT) (□) or a poly(dA)/oligo(dT) hybrid (●) to provide a homopolymeric template (dA)n. The assay was carried out in the presence of 1 mM MnCl2, 13 nM [α-32P]dTTP, Pol μ (20 ng) and 0.5 μM each DNA substrate. After incubation for the indicated times at 37°C, dTMP incorporation was quantitated as described in Materials and methods. Download figure Download PowerPoint Interestingly, the level of Pol μ-catalysed dTMP incorporation obtained on single-stranded substrates such as oligo(dT) or poly(dA), assayed independently, increased up to 370-fold when these were pre-hybridized to form a template–primer structure (Figure 4B). On the contrary, TdT catalysed a similar incorporation on both single-stranded homopolymers and a poly(dA)/oligo(dT) substrate, in agreement with its template independence (data not shown). Therefore, and in spite of its intrinsic terminal deoxynucleotidyltransferase activity, Pol μ may be defined as a DNA-dependent DNA polymerase, since it requires a template–primer for optimal activity. Pol μ is an error-prone DNA-dependent DNA polymerase When the polymerization assay on activated DNA (used to monitor Pol μ activity during purification) was carried out in the presence of all four deoxynucleotides, incorporation of the labelled dATP substrate by Pol μ was strongly inhibited (Figure 5A). In fact, under the standard conditions used to assay most DNA polymerases (>100-fold unlabelled versus labelled nucleotide precursors), Pol μ activity would not be detectable. A similar inhibition was obtained with TdT whereas, in the case of the Klenow enzyme, an increase (11-fold) in dAMP incorporation was obtained by addition of all four nucleotide substrates, as expected. Moreover, Pol μ-catalysed dAMP incorporation on poly(dT)/oligo(dA) was also inhibited strongly by relatively low concentrations of any of the other three (non-complementary) nucleotides (Figure 5B). An identical behaviour was obtained by using TdT in a parallel assay (results not shown). On the contrary, non-complementary nucleotides did not inhibit polymerization by the Klenow enzyme (see Figure 5B). These results suggest that dAMP incorporation by Pol μ is being competed by the other nucleotides, as would be expected either for a template-independent terminal transferase such as TdT, or for a DNA-dependent DNA polymerase with a poor template-directed nucleotide discrimination. Figure 5.Inhibition of DNA-directed synthesis by non-complementary dNTPs. (A) Inhibition of [α-32P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl2(a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. (B) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl2, 13 nM [α-32P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP. Download figure Download PowerPoint The ability of Pol μ to select among the four deoxynucleotides (base discrimination) to catalyse faithful template-directed DNA synthesis was evaluated initially on the four template–primer structures depicted in Figure 6, obtained as described in Materials and methods. The four dNTPs, at varying concentrations, were assayed individually as a substrate for each of the four template–primer structures, thus representing the 16 possible template–substrate nucleotide pairs (four matched + 12 mismatched). The same primer molecule (without any template) was assayed in parallel to estimate the residual terminal transferase activity of Pol μ with each of the four dNTPs. As shown in Figure 6, under the conditions used in this experiment, only dTMP was incorporated, with either Mg2+ or Mn2+, into the single-stranded DNA primer. On the contrary, on the four template–primer structures, preferential insertion of the nucleotide complementary to the first template base was observed in the presence of Mg2+ ions, indicating that the catalytic efficiency (Kcat/Km) was improved greatly by template selection of the incoming nucleotide (note that the complementary nucleotide is provided at a 10-fold lower concentration than that used in the control without template). However, template instructions appear not to be very rigorous, since the enzyme is able to add non-complementary nucleotides at 100 μM (dT and dA in Figure 6), and non-complementary dCTP at higher concentrations (data not shown). The probability of G:A misincorporation was estimated to be only 10- to 50-fold lower than that of a correct G:C pair (data not shown). Considering the efficiency of dTMP incorporation on single-stranded DNA, it cannot be ruled out that the observed insertion of dTMP using non-complementary templates might be due to residual amounts of non-hybridized primer. Figure 6.Pol μ-catalysed misinsertion at the four template bases. The four template–primer structures used, which differ only in the first template base (outlined), are indicated on the left. The single-stranded oligonucleotide corresponding to the primer strand was assayed in parallel as a control of DNA-independent nucleotide insertion. Mg2+-activated nucleotide insertion on each 5′-labelled DNA substrate (3.2 nM) was analysed in the presence of either the complementary nucleotide (10 μM) or each of the three incorrect dNTPs (100 μM), as described in Materials and methods. Mn2+-activated nucleotide insertion was assayed with each of the four dNTPs (0.1 μM). After incubation for 15 min at 30°C in the presence of 20 ng of human Pol μ, extension of the 5′-labelled (*) strand was analysed by electrophoresis in an 8 M urea–20% polyacrylamide gel and autoradiography. Download figure Download PowerPoint In the presence of Mn2+ as metal activator, the pattern and efficiency of nucleotide incorporation changed drastically (see Figure 6). In all cases, dNTP incorporation was driven by the presence of a template DNA (note that the nucleotide concentration was 1000-fold lower than that used for Mg2+ activation), but with a poor or null base selectivity. As an example, when dC is the first template base, the four dNTPs appear to have similar probabilities of being inserted. Exceptionally, dGTP incorporation occurred mainly in front of its complementary nucleotide. Moreover, inserted errors are elongated efficiently, favoured not only by complementarity but also as reiterative misinsertions, particularly when using dTTP and dATP substrates. In the same assay, a Pol β-like enzyme of only 20 kDa (ASFV Pol X) was shown to extend the four template–primer structures by adding only the correct (complementary) deoxynucleotide, but not by adding an excess (400 μM) of each of the three incorrect (non-complementary) deoxynucleotides (Oliveros et al., 1997). Similar results were obtained when Mn2+ was used instead of Mg2+ as metal activator (results not shown). All these results demonstrate that Pol μ is an error-prone DNA-dependent DNA polymerase. Interestingly, in the presence of its preferred activator (Mn2+), Pol μ behaves as a strong mutator, lacking base discrimination during nucleotide insertion on a DNA template–primer structure. Exceptionally, Pol μ preferentially inserts a dG in front of its complementary dC template base even in the presence of Mn2+ ions. Pol μ mRNA is expressed predominantly in peripheral lymphoid tissues Quantitative analyses of Pol μ transcription levels in different human tissues were carried out by Northern blotting using commercial membranes containing normalized amounts of poly(A)+ RNA from different human tissues (see Materials and methods). As shown in Figure 7, a major transcript migrating at ∼2.6 kb, in agreement with the size of the cDNA isolated (2589 nucleotides), was accumulated at the highest level in lymph nodes, followed by spleen, thymus, pancreas and peripheral blood lymphocytes. Lower levels of
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