Human DNA polymerase mu (Pol µ) exhibits an unusual replication slippage ability at AAF lesion

Abstract

We analyzed the ability of various cell extracts to extend a radiolabeled primer past an N-2-acetylaminofluorene (AAF) adduct located on a primed single-stranded template. When the 3′ end of the primer is located opposite the lesion, partially fractionated human primary fibroblast extracts efficiently catalyzed primer-terminus extension by adding a ladder of about 15 dGMPs, in an apparently non-templated reaction. This activity was not detected in SV40-transformed fibroblasts or in HeLa cell extracts unless purified human DNA polymerase mu (Pol µ) was added. In contrast, purified human Pol µ alone could only add three dGMPs as predicted from the sequence of the template. These results suggest that a cofactor(s) present in cellular extracts modifies Pol µ activity. The production of the dGMP ladder at the primer terminus located opposite the AAF adduct reveals an unusual ability of Pol µ (in conjunction with its cofactor) to perform DNA synthesis from a slipped intermediate containing several unpaired bases.

INTRODUCTION

Recently, a systematic screening of human sequence databases allowed the identification of DNA polymerase mu (Pol µ) (1,2). This novel DNA polymerase belongs to the X-family, whose members are structurally related to Pol β, a non-processive enzyme with no proofreading activity that is involved primarily in base excision repair. In vertebrates, the X-family includes also terminal deoxynucleotidyltransferase (TdT), a DNA-independent DNA polymerase whose expression is restricted to primary lymphoid tissues, and the recently identified Pol λ, predicted to be involved in DNA repair synthesis during meiotic recombination (3) and base excision repair (4). The N-terminal portion of Pol µ, Pol λ and TdT contain a BRCT motif (identified initially at the C-terminal domain of BRCA1 protein) that have no counterpart in Pol β (1–3). This BRCT motif is proposed to mediate protein–protein or DNA–protein interactions (5,6) and may specifically modulate the activity of these homologous DNA polymerases, thereby defining their biological function.

Preferential expression of Pol µ in secondary lymphoid organs led to the hypothesis of a specific involvement of this error-prone DNA polymerase in somatic hypermutation of the V regions of immunoglobulin genes (2,7). However, recent data indicate that a combination of several error-prone DNA polymerases might contribute to this process: Pol ζ (8), Pol η and Pol κ (9,10) as well as Pol ι (11) are potential candidates (for a review on new eucaryotic DNA polymerase, see ref. 12). Further investigations are required to determine whether Pol µ is also involved in this process.

Pol µ has also been proposed to be the DNA polymerase responsible for DNA synthesis during the repair of double-strand breaks by non-homologous end-joining (NHEJ) (7,13). NHEJ is the predominant form of repair of double-strand breaks occurring in vertebrate mitotic cells exposed to oxidation or ionizing radiation, particularly in the G₁ phase (14,15). NHEJ is  also an essential mechanism that repairs DNA segments during V(D)J recombination of the immunoglobulin and T cell-receptor genes. During this process, TdT contributes to antigen-receptor diversity by adding nucleotides to DNA ends. The interaction of TdT with the Ku70/86 heterodimer that binds to DNA ends may target the enzyme to the recombination sites (16). In agreement with the structural similarity of Pol µ and TdT (41% overall amino acid identity), it has been speculated that Pol µ is also able to interact with the Ku subunits and play a general role in NHEJ (7). The potential role of the DNA polymerases that could be involved in this process in mammalian cells has been investigated recently (17). In yeast Saccharomyces cerevisiae, the pol β-related POL IV was found to be required for NHEJ (18).

Recent data show that human Pol µ is able to efficiently extend mismatched bases by annealing the primer end with a microhomology region in the template 3 or 4 nt downstream (13). In the work reported here, we present evidence that Pol µ in conjunction with additional factor(s) present in the cellular extract, and most likely recruited at an N-2-acetylaminofluorene (AAF)-adduct lesion, is able to perform DNA synthesis from a slipped intermediate containing several looped-out bases. This property of promoting DNA synthesis despite the presence of mismatched nucleotides near the primer terminus is also consistent with a role in processing DNA ends during NHEJ.

MATERIALS AND METHODS

Cell lines and culture conditions

Human primary fibroblast cell strains were derived from the skin of normal (1BR) or XPV (XP6DU) individuals (19). SV40-transformed human fibroblasts [MRC5 (20) or CTAg (21)] and HeLa cells were also used.

Cells were grown at 37°C, 5% CO₂ in Dulbecco’s modified Eagle’s medium (Gibco-BRL) supplemented with 15% fetal calf serum (Eurobio) and gentamycine (50 µg/ml; Gibco-BRL).

Preparation of cell extract

Total cell extract was prepared by homogenization of the cells in hypotonic buffer as described elsewhere (19). Fraction designated as fraction B was recovered in a 30–66% ammonium sulfate cut, dialyzed and stored at –70°C. Protein concentration was determined by Bradford protein assay (Bio-Rad) using bovine serum albumin as a standard.

Primer extension assay

Construction of single-stranded plasmid with a unique AAF adduct, pUC3G1.ss, has been extensively described previously (22). The site-specific modified template was mixed with a 5′-³²P-labeled primer (5′-AGTGCCAAGCTTAGTCGATGTCCC-3′) whose 3′ end is opposite to the lesion (see Fig. 1A). That primer is protected in 3′ and 5′ with phosphorothioate bonds to prevent nucleotidyl degradation by exonuclease activities in the cellular extracts. Annealing of the primer was achieved by heating the template/primer mixture (ratio 4/1) to 70°C and allowing it to cool to room temperature for several hours. The annealed template/primer (3 fmol) was incubated in the presence of 5 µg of cell extracts in a 2 µl reaction volume containing 50 mM HEPES, 7 mM MgCl₂, 1 mM DTT and 1 mM dNTPs. When indicated, 1 ng of purified Pol µ supplemented with 5 µg of BSA was used instead of the cell extracts. After 30 min at 37°C the reaction was stopped by addition of 2 µl of stop mix (100% formamide, 10 mg/ml xylene blue, 10 mg/ml bromophenol blue). ³²P-labeled products were analyzed by electrophoresis through 20% polyacrylamide/7 M urea gels. Radiolabeled incorporation was visualized by phosphorimaging (Molecular Dynamics). For data presentation, gels were edited with Adobe Photoshop 5.5. Human purified Pol µ was purified as described previously (2).

Figure 1.

LT extension reaction catalyzed by various cell extracts. (A) The single-stranded DNA template, pUC3G1.ss, containing a single AAF adduct was annealed to a 5′-³²P-labeled primer whose 3′ end was opposite to the lesion. (B) Primer extension catalyzed by 5 µg of cell extracts (fraction B) in the presence of 1 mM dGTP were analyzed by electrophoresis through a 20% polyacrylamide/7 M urea gel.

Purification of human Pol µ

Escherichia coli cells expressing human Pol µ, obtained as described (2), were grounded with alumina for 20 min at 4°C, the resulting lysate was resuspended in buffer A (50 mM Tris–HCl pH 7.5, 1 mM DTT, 1 mM EDTA, 4% glycerol, 0.1 mg/ml BSA) supplemented with 0.5 M NaCl (6 vol/g of cells), and centrifuged for 15 min at 15 000 g to separate alumina and insoluble proteins (debris) from the soluble extract. All the following purification steps were carried out at 4°C. The supernatant was diluted with buffer A + 0.5 M NaCl to reach 120 OD₂₆₀ U/ml, and the DNA present in the soluble extract was removed by polyethyleneimine (PEI) precipitation. By adding 0.3% PEI (10% stock solution, in water, at pH 7.5) and stirring for 10 min, DNA forms a white precipitate that sediments by centrifugation at 15 000 g for 20 min. The resulting supernatant was treated with ammonium sulfate at 50% saturation to obtain a PEI-free precipitate containing most of the Pol µ. Afterwards, this precipitate was resuspended in buffer A + 50 mM NaCl and loaded in a phosphocellulose column equilibrated in the same buffer. Pol µ eluted at an ionic strength corresponding to 0.3–0.5 M NaCl. This Pol µ-enriched fraction was diluted in buffer A up to 0.3 M NaCl, loaded in a HiTrap heparin column (Pharmacia Biotech) and eluted at 0.4 M NaCl. The final fraction contained highly purified Pol µ (>95%) in soluble form. To assess that Pol µ is the only DNA polymerase present, the final heparin–Sepharose fraction was loaded onto a 5 ml glycerol gradient (15–30%) containing 20 mM Tris–HCl pH 8, 200 mM NaCl, 1 mM EDTA and 1 mM DTT, and centrifuged at 62 000 r.p.m. (Beckman SW.50 rotor) for 24 h at 4°C. After centrifugation, 20 fractions were collected from the bottom of the tube, examined in Coomassie Blue-stained gels and tested for DNA polymerase activity on activated DNA. A single peak of DNA polymerase activity perfectly co-sedimented with the Pol µ (∼55 kDa) polypeptide.

Obtention of polyclonal antibodies specifically recognizing human Pol µ

Rabbit polyclonal antibodies specific for Pol µ were developed via innoculation of the complete human Pol µ enzyme overproduced in E.coli cells (300 µg). The IgG fraction was purified by chromatography on protein A–Sepharose (Bio-Rad), dialyzed for 2 h against 30 mM HEPES, 1 mM DTT, 100 mM glutamic acid and 10% glycerol, and stored at –70°C. The sensitivity of the rabbit antisera was tested by dot blotting, using different amounts of purified Pol µ as antigen. (The antibody, at a dilution of 1:10.000, was able to detect 50 pg of purified Pol µ using the ECL detection system.) The specificity of the Pol µ antibodies was confirmed by western blotting of different protein extracts, and also by dot blotting. Using this method, cross-reactivity with Pol λ was estimated to be <1%.

Real-time quantitative RT–PCR

RNA was extracted from the cell cultures using Trizol reagent (Gibco-BRL). RNAs were resuspended at 1 µg/µl in H₂O-DEPC containing 2 U/µl of SUPERase•IN (Ambion). In order to prevent the presence of contaminating DNA, RNAs were treated with RQ1 RNase-free DNase (1 U/7 µg RNA; Promega). After 15 min incubation at 37°C, the reaction was stopped by adding 10 mM EGTA. RNAs were precipitated and resuspended at 1 µg/µl in H₂O-DEPC.

Total RNA was reverse transcribed with the RETROscript™ Kit (Ambion) using random decamers primers. Variable amounts of cDNA were amplified by PCR in a reaction mixture (20 µl) that contained 2.5 mM MgCl₂, 0.25 µM of each primer and 2 µl of LC FastStart DNA Master Mix SYBR Green I (Roche Diagnostics). The primers (OligoExpress) used for the amplification were as follows: Pol µ forward, 5′-AGGCTGTCGTCCCAATGCTC-3′ (located in exon 1); Pol µ reverse, 5′-CAGGCATAGGCAGGCATCCA-3′ (located in exon 3); GAPDH forward, 5′-GTTCGACAGTCAGCCGCATC-3′ (located in the 5′ untranslated region); and GAPDH reverse, 5′-TTGAGGCTGTTGTCATACTTCTCAT-3′ (located in exon 6). The following LightCycler run program was used: denaturation step (95°C for 10 min), amplification step 45 times (95°C for 15 s, 60°C for 10 s, 72°C for 20 s) with a single fluorescence measurement per cycle, melting step (95°C for 10 s, 72°C for 10 s, 98°C with a heating rate of 0.1°C per s) with a continuous fluorescence measurement. Melting curve analysis and electrophoresis on a 1.8% Metaphor agarose gel (FMC Bioproducts) were performed to check the T_m and the size of the PCR products.

For a mathematical analysis of the quantitative RT–PCR, we determined the crossing points (CP) by the second derivative maximum method in the LightCycler 3.5 software (23). The CP is the point where the fluorescence rises above the background signal, indicating the beginning of the exponential phase of the PCR. Log cDNA quantity was plotted against CP. The efficiency (E) of amplification for each cDNA was calculated according to the equation E = 10^[–1/slope]. CP deviation (ΔCP) of each strain (sample) versus 1BR cells (control) was determined. Relative expression (R) of Pol µ transcript was calculated by using the equation 1. GAPDH transcript was used as a reference. The results are expressed as a percentage of Pol µ mRNA content in 1BR cells.

1

RESULTS AND DISCUSSION

Detection of a DNA polymerase activity that efficiently extends a lesion terminus (LT) by adding a ladder of nucleotides

In the course of experiments aimed at understanding the role of various proteins in the extension step of translesion synthesis, we developed a primer elongation assay using a specific template/primer containing a single AAF adduct. The modified G-AAF single-stranded template was annealed to a primer whose 3′ end is located opposite the lesion (Fig. 1A) thus forming a replication intermediate referred to as the LT. Upon incubation of the template/primer with cellular extracts in the presence of dGTP only, incorporation of three dG residues was expected (at the most) given the template sequence context of three dC residues located 5′ to the AAF adduct. Surprisingly, a specific fraction (fraction B, see Materials and Methods) from normal (1BR) or XP6DU primary fibroblasts extracts extended very efficiently the LT, by adding a ladder of about 15 dGMP residues (Fig. 1B). In contrast, incorporation of dGMP was reduced in equivalent extracts from MRC5, CTAg and HeLa cells (Fig. 1B).

Normal and XP6DU (deficient in Pol η) (24,25) fibroblasts showed the same pattern of elongation, clearly indicating that the dG ladder-synthesizing activity is not mediated by DNA Pol η but by another DNA polymerase that remained to be identified.

The presence of AAF adduct triggers the dG ladder-synthesizing activity

In order to characterize further the enzymatic properties of the fractionated 1BR extract, its ability to elongate an oligonucleotide in the absence of a template was investigated. As shown in Figure 2A, no elongation could be detected with any of the nucleotides tested. Conversely, in the presence of the corresponding non-modified template and dGTP, three guanine residues were incorporated, as predicted by the sequence context (Fig. 2B). Only a faint signal of incorporation was detected when the other three dNTPs were assayed individually. In these cases, the mobility of the extended products is identical to the one of the dG-extended products, suggesting that they correspond to accurate elongation mediated by low endogenous levels of dGTP present in the extract. As an exception, by adding dCTP, a ‘true’ dCMP-extended product is observed at the +1 position.

Figure 2.

The presence of AAF adduct on the DNA template triggers the dG ladder-synthesizing activity. Analysis of primer extension catalyzed by 5 µg of 1BR protein extract (fraction B) in the absence (0) or presence of a single deoxyribonucleoside triphosphate, dATP (A), dCTP (C), dGTP (G) or dTTP (T), or the four dNTPs together (4). The assay was carried out comparatively on either a single primer (A), a non-modified pUC3G0.ss/primer (B) or a pUC3G1.ss/primer (C and D).

In the presence of the AAF-modified substrate, 1BR extract catalyzed the aberrant primer extension of the LT by using exclusively dGTP (Fig. 2C). The extension pattern obtained in the presence of all four dNTPs was identical, suggesting that, in this particular sequence context, dGTP is the only nucleotide selected even when a pool of the four dNTPs is provided (Fig. 2D).

Taken together, these results indicate that fraction B of human primary fibroblasts contains a DNA-dependent polymerase that displays a dG ladder-synthesizing activity, only in the presence of the AAF adduct. This aberrant reaction may be triggered by the strong distortion that a G-AAF adduct imposes upon the DNA template (26). The specificity and reiteration of the end-addition reaction being restricted to dGMP suggest that it is driven by the run of three Cs downstream to the AAF adduct on the template strand.

Pol µ complements deficient extracts

The observations mentioned above are consistent with the possibility that the LT extension observed when using extracts from fibroblasts is mediated by a DNA polymerase related to TdT. However, as TdT expression is restricted to primary lymphoid cells at a precise stage of their differentiation, it is unlikely that the observed LT extension in fibroblast extracts is due to TdT. We decided to test Pol µ, which shares a 41% amino acid identity with TdT. In vitro, the purified enzyme displays intrinsic terminal deoxynucleotidyl transferase activity but, unlike TdT, the catalytic efficiency of polymerization carried out by Pol µ was strongly enhanced by the presence of a template strand (2). These biochemical properties of Pol µ were tested using the same template/primer structures as above. As shown in Figure 3A, 1 ng of purified Pol µ was able to catalyze the addition of one dTMP to a DNA primer in the absence of a template. This nucleotide preference has been described previously (2). In the presence of the non-modified primer/template substrate, template-directed insertion of three dGMPs occurred. The enzyme was also able to add dTMP and dCMP but less efficiently than dGMP (Fig. 3B). These observations are consistent with the biochemical properties of Pol µ, including an optimal activity in the presence of a template/primer and low fidelity (2). When the reaction was carried out using the AAF mono-modified template (Fig. 3C), Pol µ is able to catalyze template-directed addition of three dGMPs more efficiently than misincorporation of dTMP and dCMP. Besides, the primer appears to be more efficiently elongated (number of primers extended), but the proportion of primer molecules stalled at position +2 in the presence of dGTP is higher when using the AAF mono-modified template. The extension catalyzed by Pol µ stops after three dGMPs have been incorporated even though all the four dNTPs are included in the reaction, suggesting that the presence of the AAF adduct hinders further progression of the DNA polymerase (Fig. 3D). A similar inhibition of elongation can be observed when dTMP and dCMP are independently provided.

Figure 3.

Pol µ-dependent primer extension. The assay was carried out comparatively with either a single primer (A), a non-modified pUC3G0.ss/primer (B) or a pUC3G1.ss/primer (C–G), in the absence (0) or presence of a single deoxyribonucleoside triphosphate, dATP (A), dCTP (C), dGTP (G) or dTTP (T), or the four dNTPs together (4), as indicated. Reactions were performed by using 1 ng of purified Pol µ in the presence of either 5 µg of BSA (A–D) or 5 µg of a HeLa total extract (E and F). (G) The activity of 5 µg of HeLa total extract in the absence of Pol µ.

A dramatically different result was obtained when Pol µ activity was assayed in the presence of a cellular extract that was defective in the dG ladder-synthesizing activity, such as a HeLa total extract (Fig. 3E). In this case, the specificity of the nucleotide insertion was very strict, and no other dNTP but dGTP could be incorporated. Moreover, a run of about 15 dGMPs was incorporated instead of the three dGMP residues obtained when purified Pol µ was used alone. This extension pattern was identical to that observed by using primary fibroblast extracts.

The ability of Pol µ to complement a deficient extract suggests that a cofactor(s) present in the extract modulates the activity of the DNA polymerase, modifying its incorporation pattern. This cofactor(s) is specific for Pol µ, as purified Pol β, although being as efficient as Pol µ for the extension of the LT by three dGMPs failed to restore the dG ladder-synthesizing activity in the deficient extract (data not shown). These results demonstrate that Pol µ and Pol β are not interchangeable and that the dG ladder-synthesizing activity specifically results from an interaction between Pol µ and cofactor(s) present in the extracts.

Anti-Pol µ antibodies specifically inhibit the dG ladder-synthesizing activity

To further confirm that the dG ladder-synthesizing activity can be attributed to Pol µ, LT extension was assayed in the presence of either native or heat-denatured antibodies raised against human Pol µ. Native antibodies inhibited the catalytic activity of purified Pol µ (Fig. 4A). The level of inhibition quantified by phosphorimagery varied from 14- to 5-fold depending to the concentration of IgG used. LT extension catalyzed by a primary fibroblast extract (1BR; Fig. 4C), as well as that of a reconstituted extract (MRC5 extract supplemented with Pol µ; Fig. 4B), were also inhibited by the antibody in a dose-dependent manner. Inhibition was not observed with an unrelated antibody (data not shown) or when using heat-inactivated anti-Pol µ antibodies (Fig. 4). The data provided evidence that Pol µ is indeed the DNA polymerase responsible for the observed dG-ladder formation. Furthermore, IgG anti-Pol µ failed to inhibit DNA polymerase activity of Pol β (Fig. 4D) showing that the antibody specific for DNA polymerase µ has no effect on other polymerase.

Figure 4.

IgG anti-Pol µ inhibits specifically LT extension. The assay was carried out using the pUC3G1.ss/primer and 1 mM dGTP. A range of IgG anti-Pol µ antibodies (375 or 187 ng/µl), either native or heat denatured (92°C for 10 min), was included in the reaction as indicated. Reactions were performed by using 1 ng of purified Pol µ in the presence of 5 µg of BSA (A), or in the presence of 5 µg of a MRC5 total extract (B). The activity of 1BR extract (fraction B) is shown (C) with 375, 187 or 75 ng/µl of IgG anti-Pol µ antibodies. (D) pUC3G0.ss/primer (without lesion) is incubated with a range of Pol β (100, 10 or 1 pg) in the presence of 5 µg of BSA and 1 mM dGTP. IgG anti-Pol µ antibodies (375 ng/µl) were included in the reaction as indicated

Expression of Pol µ mRNA correlates with the dG ladder-synthesizing activity

The LT primer elongation assay used in this study allowed a clear distinction between primary cell extracts which displayed an efficient dG-ladder formation and transformed cell extracts which hardly incorporate >4 nt. In order to compare the expression of Pol µ in these different cell strains, real-time quantitative RT–PCR was performed using serially diluted cDNA. Amplification of the GAPDH gene was used to normalize the results. The specificity of the PCR products was documented by electrophoresis and T_m determination. Cycle number of CP versus cDNA input were plotted to calculate the slope (Fig. 5A–C). The average PCR efficiencies using the cDNA from the various cell lines were identical for GAPDH (1.87 ± 0.06) and for Pol µ (1.87 ± 0.15). By using the established mathematical model of Pfaffl (23), we determined that Pol µ expression in XP6DU cells was almost identical to that observed in 1BR cells (66.7%). In contrast, Pol µ expression was significantly down regulated in CTAg (13.7%), MRC5 (4%) and HeLa cells (2.6%). Thus, modulation of Pol µ mRNA expression correlates with the detection of LT elongation in cell extracts, which is consistent with the implication of Pol µ in this process.

Figure 5.

Expression of Pol µ mRNA correlates with the dG ladder-synthesizing activity. Determination of real-time RT–PCR efficiencies of target gene (Pol µ) and reference gene (GAPDH). CP cycles versus log 10 of cDNA input were plotted for 1BR (A), MRC5 (B) and XP6DU cells (C) to determine the slope. The corresponding real-time RT–PCR efficiencies were calculated according to the equation E = 10^[–1/slope]. Relative ratio of Pol µ cDNA in the different strains versus 1BR cells was determined according to equation 1 (see Materials and Methods). The results are expressed as a percentage of Pol µ mRNA content in 1BR cells (D).

CONCLUSION

We show that the Pol µ present in human primary fibroblast extracts is able to extend efficiently the LT formed by a primer whose 3′ end anneals opposite an AAF adduct. The purified enzyme was unable per se to extend the primer by >3 nt. In marked contrast, in the presence of a cellular extract, Pol µ efficiently catalyzed LT extension by producing a ladder of about 15 dGMPs. It is unlikely that the TdT-like activity of DNA Pol µ could be involved in the production of the dG ladder. Indeed it has been shown that DNA Pol µ is able to catalyze polymerization of any of the four dNTPs to a single-stranded DNA primer in the absence of a template, dTTP and dCTP being inserted more efficiently. We prefer to propose the following model (Fig. 6): (i) Pol µ extends the primer with one, two or three dGMPs; (ii) incorporation of additional nucleotides appears to be impeded by the presence of the AAF adduct in the primer binding cleft of the DNA polymerase; (iii) asymmetric translocation of the primer strand (slippage) repositions its 3′ end such that further extension by Pol µ occurs again opposite the dC repeat; (iv) several cycles of slippage/extension produce a loop of unpaired bases on the neosynthesized strand; (v) the process stops, probably when the bulge in the primer strand gets too big to support stably DNA polymerase binding.

Figure 6.

Schematic representation of LT extension by Pol µ in the presence of a cellular extract. (A) Extension of the LT by Pol µ which incorporates three dGMPs. (B) AAF adduct hinders further Pol µ progression. (C) Reannealing of the primer terminus takes place by a slippage mechanism involving the 3C repeat. (D) The polymerase goes forward and elongates the primer until it will be blocked again by the AAF adduct. (E) Several cycles of sliding back/elongation increases the size of the neosynthesized strand containing unpaired bases.

The entire process is strictly dependent upon the presence of a cellular extract that provides a cofactor(s) for dG-ladder formation. We propose that, by means of either a post-translational modification of Pol µ or a protein–protein interaction, the cofactor may allow the DNA polymerase to be more tightly associated with the DNA despite the presence of several unpaired bases. Alternatively, the slippage reaction may occur upon DNA polymerase dissociation from the template after pausing at the AAF site. In this case, the cofactor may facilitate the remodeling of the substrate and reloading of the polymerase to the distorted primer terminus.

The assay described here provides an unique opportunity to identify the cofactor(s) that is likely to have an important regulatory role of the biological function of Pol µ. DNA-PK modifies TdT activity in vitro by limiting both the length and composition of nucleotide addition (27). As TdT and Pol µ share a 41% overall amino acid identity in both their BRCT and Pol β core domains, we tested the influence of DNA-PK on Pol µ activity. Until now, we failed to demonstrate any functional interactions between the purified proteins to produce the dG ladder.

Pol µ has the peculiar capacity of accepting distortions in both primer and template strands, being able to extend realigned mismatched primer terminus, and to produce misinsertions on the same basis (13). Such a feature, together with the biochemical properties reported in this paper, could be especially useful for the proposed function of Pol µ in NHEJ (7). This DNA polymerase could produce insertions or deletions resulting from template/primer misalignments due to replication blockage by either a secondary structure or a chemical lesion.