Fig. 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.

Fig. 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).

Fig. 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.

Fig. 7. Pol μ mRNA is expressed preferentially in secondary lymphoid organs. Northern blotting analysis of TdT-2 mRNA was carried out as indicated in Materials and methods, using commercial blots (MTN and MTN–II blots, Clontech) containing poly(A)⁺ RNA from the human tissues indicated. The membrane was hybridized with a specific ³²P-labelled DNA probe containing 1141 nucleotides of the Pol μ cDNA 3′–terminal sequence. The hybridized probe, revealing a major transcript (2.6 kb), was detected by autoradiography.

Fig. 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).

Fig. 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.

Fig. 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.

Fig. 8. In situ hybridization of Pol μ mRNA in different human tissues. DIG-labelled sense and antisense riboprobes, corresponding to the first 1200 nucleotides of Pol μ cDNA, were obtained and hybridized to human tissue sections (Human Tissue Set I and Human Hematal and Immune Tissue Set, Novagen) under the conditions described in Materials and methods. After hybridization, detection of the RNA probes in tissue sections was carried out by incubation with anti-DIG–alkaline phosphatase antibody.The dark blue staining, observed in lymph nodes and spleen with the antisense riboprobe, outlined regions largely expressing Pol μ mRNA. No comparable signal was obtained by using a sense riboprobe under the same experimental conditions and in a close parallel tissue section.