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. 2002 Apr 1;30(7):1493–1499. doi: 10.1093/nar/30.7.1493

Physical and functional interactions of the tumor suppressor protein p53 and DNA polymerase α-primase

Christian Melle 1, Heinz-Peter Nasheuer 1,a
PMCID: PMC101832  PMID: 11917009

Abstract

The wild-type form of p53 contains an intrinsic 3′–5′-exonuclease activity. As p53 forms a complex with DNA polymerase α-primase (pol-prim) in vivo this finding suggests that p53 might cooperate with pol-prim to stabilize the genetic information of living cells. To test this hypothesis, exonuclease-free DNA pol-prim was expressed alone or together with p53 for purification. Pol-prim formed a complex with p53, which was purified by ion exchange and immunoaffinity chromatography from baculovirus-infected insect cells. The p53-containing pol-prim fractions removed a 3′-unpaired nucleotide with a 1.5–2-fold higher rate than a paired nucleotide, whereas the four subunit pol-prim did not have any exonuclase activity. Therefore, only p53/pol-prim was able to elongate a primer-template that contained a 3′-unpaired primer end in vitro. To achieve this, the 3′–5′-exonuclease activity of p53 excised the unpaired nucleotide at the 3′-end of the primer and created a paired 3′-end, which pol-prim was able to elongate. The exonuclease activity of p53 as well as the elongation of a primer with a mispaired 3′-end was inhibited specifically by the anti-p53 monoclonal antibodies PAb240 and PAb421.

INTRODUCTION

The tumor suppressor protein p53 has multiple functions in preventing cellular growth and tumor development by conserving genetic integrity in mammalian cells (1–7). The loss or functional inactivation of p53 greatly enhances the risk of malignant transformation of cells. These findings underline its critical role in preventing cancer and indeed ∼50% of all human tumors contain mutant p53 genes (8–10). In contrast to its recently discovered homologs (11,12) and despite its central functions in cancer, p53 is not essential for the development and growth of cells (13).

Analysis of p53 functions and their regulation have led to a model for a complex network of activities (14). Wild-type p53 is involved in the regulation of the cell cycle as a transactivator of important regulatory proteins (15–18), in DNA repair by interacting with essential repair proteins (19–22) and is probably directly involved in cellular DNA replication (23), as well as in recombination events (24–27). Furthermore, it binds to DNA polymerase α-primase (pol-prim) and to the eukaryotic single-stranded DNA-binding protein, replication protein A (RPA) (28,29). In addition, it was shown that p53 contains an intrinsic 3′–5′-exonuclease activity, which might play a role in DNA replication or repair (30–32). Recently, it was shown that the p53 exonuclease activity has a proofreading capacity to enhance the fidelity of reverse transcriptase (RT) of the murine leukemia virus (MLV) and of the human immunodeficiency virus (HIV; 33,34). As two eukaryotic DNA polymerases, DNA pol-prim and DNA polymerase β, do not possess an intrinsic proofreading exonuclease (35), it was hypothesized that p53 fulfills this function (30,36–38). This possibility is underlined by the fact that several preparations of pol-prim were described that were associated with an exonuclease activity (39–41). Recently, a direct interaction of DNA polymerase β and p53 was shown that is responsible for increased base excision repair activity (42).

Eukaryotic DNA replication is a highly accurate process that results in error rates of ∼10–10 misincorporated nucleotides per base and cell doubling (43). Such a high accuracy is achieved by a mechanism consisting of a minimum of three steps, which are the nucleotide selection of the replicative DNA polymerase, proofreading and post-replicative mismatch repair (MMR). A decrease in the fidelity of DNA replication would lead to genomic instability and result in an increased risk of cancer (44,45). It was shown that even in the early stages of the cellular transformation process high incidences of mutations occur (46), which might well be caused by misinsertion and proofreading defects of replicative DNA polymerases. In analogy to defects of MMR genes, such defects are expected to contribute considerably to the formation and propagation of cancers. Taking these findings into account, the exonuclease activity of p53 might contribute a proofreading function to cellular DNA polymerases and thereby increase the fidelity of the cellular DNA synthesis process.

The present study was conducted to characterize the interaction of pol-prim and p53 and to investigate in this respect the intrinsic 3′–5′-exonuclease activity of p53 in vitro. This study shows for the first time the existence in vivo of a complex of pol-prim and p53 as identified by immunoprecipitation and the interplay of the DNA polymerase with the 3′–5′-exonuclease activity of the p53/pol-prim complex. Preparations of pol-prim containing p53 degraded preferentially unpaired versus paired 3′-ends whereas pol-prim without p53 did not have any exonuclease activity. p53/pol-prim exchanged a 3′-end mispair against the correct nucleotide, whereas pol-prim alone did not.

MATERIALS AND METHODS

Protein purification

The four subunits of pol-prim were overproduced by infection of High Five insect cells (Invitrogen, Heidelberg) with the appropriate baculoviruses (47–50). Human p53 was also expressed by infection of insect cells with recombinant baculoviruses (51), and purified as described (31). A complex consisting of pol-prim and p53 was overproduced in High Five insect cells by infection with the corresponding five baculoviruses. Pol-prim, as well as the p53/pol-prim complex, was purified by phosphocellulose chromatography and immunoaffinity chromatography using immobilized monoclonal antibody SJK 237-71 directed to the p180 subunit of pol-prim and subsequent alkaline elution (52,53).

Immunoprecipitation

The antibodies HP180-12 and PAb101 were bound to protein G–Sepharose beads and crosslinked on beads with dimethyl-pimelimidate (54–56). Crude extracts (850 µl) from human CEM cells were incubated with anti-pol-prim monoclonal HP180-12, which recognizes the large subunit p180 to immunoprecipitate protein complex for 1 h at 4°C (54). The anti-SV40 T-Ag monoclonal antibody PAb101 served as a negative control. Then the resins were washed three times with 20 mM HEPES–KOH pH 8, 25 mM KCl, 5 mM MgCl2, 0.1 mM EDTA and 0.05% NP-40. Bound proteins were subjected to 10% SDS–PAGE and detected by immunoblotting with anti-p180 monoclonal antibody HP180-7 (54) and anti-p53 polyclonal antibody Ab-7 (Calbiochem).

DNA polymerase assay

The activity of pol-prim was determined with activated calf thymus DNA as described elsewhere (57). Furthermore, DNA polymerase α activity was analyzed by denaturating gel electrophoresis. If not indicated otherwise the 16-base primer 1 (5′-GTA ACT TTT CCC AGC C-3′) corresponding to the position AB 5278–5293 of the ΦX174am16 genome, which includes the amber16 codon, was radioactively labeled at its 5′-end with T4-polynucleotide kinase (Biolabs) and [γ-32P]ATP (Amersham Bioscience). Then it was hybridized to the 63mer template oligonucleotide (5′-GTT CAA CCA GAT ATT GAA GCA GAA CGC AAA AAG AGA GAT GAG ATT TAG GCT GGG AAA AGT TAC-3′) derived from the ΦX174am16 sequence according to standard procedures (58–60). The 16-base primer 2 (5′-GTA ACT TTT CCC AGC G-3′) led to a 3′-terminal mispair when hybridized to the 63mer oligonucleotide and was treated as described for primer 1. These template-primer systems were used as substrates for pol-prim alone or p53-containing pol-prim complexes as indicated. Reaction mixtures contained 50 mM Tris–acetate, pH 7.5, 10 mM potassium acetate, 6 mM magnesium acetate, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.05 mM (each) of the four dNTPs, and the indicated concentrations of DNA substrates (57). The reaction was stopped by adding loading buffer (90% formamide, 10 mM EDTA, 0.1% bromphenol blue and 0.18% xylene cyanol FF) and heating the mixture to 90°C. Products were analyzed on 20% polyacrylamide gels containing 7 M urea followed by autoradiography using PhosphorImager screens (Amersham Bioscience) at –20°C or Biomax films (Kodak) at –80°C. PhosphorImager autoradiographies were quantified with the ImageQuant program (Amersham Bioscience).

Exonuclease assay

The exonuclease activity was analyzed with the same primer-template systems as described above. The reaction was performed in 50 mM Tris–acetate, pH 8.5, 10 mM magnesium acetate, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin and radioactively labeled primers (59). The reaction was started by adding 2–5 ng of p53 or 5–10 ng of p53-containing pol-prim at 37°C for 15 min or the time indicated. The reaction was stopped by adding loading buffer and heating to 90°C. Products were analyzed as described above.

Other techniques

Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard (61). Antibodies were either purified from hybridoma supernatants or rabbit serum by protein A–agarose chromatography (Dianova, Hamburg) as described (56). The purity of the antibodies and the other proteins was determined to be >90% by gel electrophoresis through a denaturing 10% polyacrylamide gel.

RESULTS

Immunoprecipitation of p53/pol-prim from human cells

The monoclonal antibodies HP180-12 and SJK237-71, which recognize the p180 subunit of human pol-prim, immunoprecipitated pol-prim and p53 from human crude extracts (Fig. 1A, lane 5, and data not shown). In parallel, the monoclonal antibody PAb101 did not precipitate pol α or p53 from the crude extracts (Fig. 1A, lane 3). The data suggest that p53 interacts with pol-prim in human cells.

Figure 1.

Figure 1

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Complex formation of pol-prim and p53. (A) CEM cells crude extracts (60 µg, lane 1)were incubated with anti-SV40 T-Ag monoclonal antibody PAb101 (lane 3, crosslinked antibody resin with crude extract) and anti-p180 monoclonal antibody HP180-12 (lane 5, crosslinked antibodies with crude extract). The antibody HP180-12 precipitated both p180 and p53 from CEM crude extract (lane 5). The anti-SV40 T-Ag monoclonal antibody PAb101 did not precipitate p180 or p53 (lane 3). As a control for solubilized antibodies the crosslinked antibody–protein G resins were incubating with loading buffer in parallel (lanes 2 and 4). The proteins were subjected to 10% SDS gel electrophoresis and detected by immunoblotting with anti-p180 monoclonal antibody Hp180-7 and anti-p53 polyclonal antibody Ab-7. (B) Four subunits of pol-prim, p53 and all five polypeptides were expressed in High Five insect cells and purified as described in the Materials and Methods. The proteins (240 ng of immunoaffinity-purified pol-prim, 70 ng of p53 and 400 ng of p53/pol-prim complex; lanes 1, 2 and 3, respectively) were analyzed by SDS gel electrophoresis and subsequent staining with silver as described earlier (58).

Purification of an enzyme complex consisting of pol-prim and p53 from baculovirus-infected insect cells

After co-infecting cells with recombinant baculoviruses encoding the four subunits of human pol-prim and p53, p53-containing pol-prim fractions were obtained by phosphocellulose and immunoaffinity chromatography using monoclonal antibodies directed against the largest subunit, p180, of pol-prim (Fig. 1B, lane 3). The immunoaffinity-purified p53/pol-prim complex was stable during gel filtration (data not shown). Omitting the p53-encoding baculovirus yielded a four subunit pol-prim complex (Fig. 1B, lane 1). The affinity-purified p53/pol-prim fraction displayed a purity of at least 95%, with regard to the four pol-prim subunits and the accompanying p53 (Fig. 1B, lane 3). The eluate from the immunoaffinity resin was dialyzed against storage buffer and stored at –80°C for further use. These enzyme complexes either had only DNA polymerase and primase activity (heterotetrameric pol-prim), or had DNA polymerase, primase, as well as 3′–5′-exonuclease activity (Fig. 2A).

Figure 2.

Figure 2

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Exonuclease activity of p53-associated and p53-free DNA pol-prim. (A) Increasing amounts of pol-prim with and without p53 were incubated with primer-template system. The reaction products were separated on a 20% denaturating polyacrylamide gel and detected by autoradiography. The immunopurified p53/pol-prim (17.5 and 35 ng at 37°C; lanes 1 and 2, respectively) degraded radioactively labeled DNA, whereas the same amounts of the four subunit pol-prim did not (lanes 3 and 4). Labeled primers before incubation with proteins are shown in lane M. (B) To compare the degradation of primers with a paired and an unpaired 3′-end, primer systems 1 and 2 were incubated with 10 ng of p53/pol-prim at 30°C for increasing times as indicated [lanes 1–4, paired primer (lane M) and lanes 5–8, primer with mismatched 3′-end (lane M*) incubated for 0.5, 2, 7 and 12 min, respectively]. After autoradiography the primers (lanes M and M*) and the exonuclease degradation products were quantified with the ImageQuant program. The value of the 3′-unpaired primer (lane M*) was arbitrarily set to 100. The relative value of the paired primer (lane M) was 118 whereas the relative values of the exonuclease products were 7.1, 9.7, 14, 14.7, 10.9, 14.3, 16.4 and 18 (lanes 1–8, respectively). As the intensity of the paired primer was greater than that of the 3′-unpaired primer (lanes M and M*, respectively) the values of the exonuclease products of the paired primer (lanes 1–4) were normalized to equal the radioactivity of both primers for the purpose of comparison. After 0.5, 2, 7 and 12 min the normalized values of the exonuclease products were 6.1, 8.2, 11.9 and 12.5 for the paired primer, and 10.9, 14.3, 16.4 and 18 for the unpaired primer, respectively.

When expressed alone, recombinant p53 was purified by phosphocellulose chromatography and subsequent immunoaffinity chromatography using the immobilized monoclonal antibody PAb421 directed against the C-terminus of p53 (Fig. 1B, lane 2). Both p53-containing fractions also displayed 3′–5′-exonuclease activity. The p53 subunit of the enzyme complex or p53 alone possessed similar running properties in the gel and the proteins were highly purified (Fig. 1B).

Only p53-containing pol-prim showed 3′–5′-exonuclease activity

Affinity-purified pol-prim either alone or from cells co-infected with p53-encoding baculoviruses was subjected to exonuclease assays. Only p53-containing fractions of pol-prim displayed exonuclease activity whose biochemical characteristics were identical to those of the previously purified p53 alone (Fig. 2A, lanes 1 and 2) (31). These included a 3′–5′ directionality (data not shown), a distributive rather than a processive mode of excision and a pattern of products that was indistinguishable from that obtained with p53 alone. On the other hand, pol-prim without p53, which was purified in parallel, did not display exonuclease activity (Fig. 2A, lanes 3 and 4). The exonuclease of the isolated p53/pol-prim complex excised mismatched G-G nucleotides over matched nucleotides G-C with a ratio of about 1.8:1 after 0.5 and 2 min (Fig. 2B, lanes 1, 2, 5 and 6). Further characterization revealed that the antibodies PAb240 and PAb421, but not the antibody DO-1, inhibited the p53 exonuclease activity (Fig. 3, lanes 4–6, 7–9 and 1–3, respectively).

Figure 3.

Figure 3

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Inhibition of p53 exonuclease activity by monoclonal antibodies. Increasing amounts of p53-specific monoclonal antibodies DO-1, PAb240 and PAb421 were titrated in an exonuclease assay containing equal amounts of p53/pol-prim to investigate a possible inhibitory effect of these antibodies. PAb240 (0.7 and 1.4 µg, lanes 5 and 6, respectively) and PAb421 (0.7 and 1.4 µg, lanes 8 and 9, respectively) inhibited the p53 exonuclease activity (lanes 4 and 7). DO-1 (0.7 and 1.4 µg, lanes 2 and 3, respectively) did not inhibit p53 exonuclease activity (lane 1). The products were analyzed by denaturating polyacrylamide gel electrophoresis and autoradiography.

p53-containing pol-prim but not the p53-free form of pol-prim allowed the elongation of a mispaired 3′-end

To study the cooperation of the exonuclease and DNA polymerase functions of p53/pol-prim, we conducted two synthetic 16/63mer template-primer systems, one containing a G:G mispair at its 3′-end and the other with a paired end. The latter was used efficiently as a substrate by both the pol-prim complex with and the one without p53, and both enzyme complexes had strong and comparable DNA synthesizing activities (Fig. 4A and B, lanes 1–4). In contrast, the mispaired primer was exclusively elongated by the p53-containing enzyme complex (compare Fig. 4A and B, lanes 5–8). In addition to DNA synthesis activity, p53/pol-prim shows products resulting from 3′–5′-exonuclease activity when using the mispaired primer (Fig. 4A, lanes 7 and 8). The elongation of the mispaired primer was inhibited by the p53-specific antibodies PAb421 and PAb240 but not by heat-denaturated antibodies or by the p53-specific antibody DO-1 (Fig. 5, compare lanes 1–3 with lanes 4–6; data not shown). This inhibition of the elongation reaction was most probably caused by abolishing the p53 exonuclease activity (Fig. 3). These findings strongly suggest that the p53-associated exonuclease from the p53/pol-prim complex was mandatory for the subsequent elongation of the mispaired primer by DNA polymerase α.

Figure 4.

Figure 4

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DNA synthesis of p53-associated and p53-free DNA pol-prim on different primer-template systems. The ability of p53-associated (A) and p53-free pol-prim complexes (B) to extend either a paired or a mispaired 3′-end primer-template system was compared. Increasing amounts of proteins were incubated with the primer-template system 1 (paired 3′-end, lane M without pol-prim, and lanes 1–4 in the presence of 0.09–0.9 DNA polymerase units of each enzyme complex) and system 2 (mispaired 3′-end, lane M* without pol-prim and lanes 5–8 in the presence of 0.09–0.9 DNA polymerase units of each enzyme complex) for 30 min at 37°C. The products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography.

Figure 5.

Figure 5

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The elongation of a mispaired 3′-end by p53/pol-prim complex is inhibited by PAb421. To determine whether the elongation of a mispaired 3′-end primer depends on the exonuclease activity of p53 either untreated (0.7 and 1.4 µg, lanes 2 and 3, respectively) or heat-denatured (0.7 and 1.4 µg, lanes 5 and 6, respectively) purified PAb421 was added to the reaction mixture. Primer system 2 was incubated with 0.2 DNA polymerase units of p53/pol-prim for 30 min at 37°C in the presence (lanes 2, 3, 5 and 6) or absence (lanes 1 and 4) of antibody. The products were analyzed by denaturating polyacrylamide gel electrophoresis and autoradiography.

To study the exchange of a mispaired base the incorporation of the correct nucleotide and another mispaired nucleotide was investigated. During incubation of the mispaired 3′-end in the presence of the correct dNTP, p53-containing pol-prim removed the incorrect dGMP and replaced it with the correctly paired dCMP (Fig. 6B, lanes 6–10). Such a replacement reaction was not detected with pol-prim alone and no products were detected (data not shown). In contrast, p53-containing pol-prim did not detectably incorporate an incorrect dNMP, such as dAMP, at the 3′-end instead of the mispaired dGMP (Fig. 6C, lanes 11–15). It is worth mentioning that the replacement of an incorrect nucleotide was rather slow (Fig. 6B, lanes 6–10). Elongation of a correctly paired primer was 50% complete within 30 s (Fig. 6A, lanes 1–5) whereas significant amounts of product from the exchange reaction were only detectable after 5–10 min (Fig. 6B, lanes 6–10). The data suggest that the exonuclease was too slow, that additional factors such as RPA were missing in the assay system, or that specific modifications might be required for an optimal cooperation of pol-prim and the p53 exonuclease activity. For this reason the effects of RPA on this nucleotide exchange assay were examined. A reproducible, 1.5–2-fold increased rate of DNA synthesis by p53/pol-prim on mispaired primer-template was observed in the presence of RPA (data not shown). This indicates that additional factors or modifications may be necessary for a satisfactory rate of DNA synthesis in this type of assay.

Figure 6.

Figure 6

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Exchange of a 3′-unpaired nucleotide by p53-associated DNA pol-prim. Unlabeled primer-template systems containing either a paired (primer 1, A) or mispaired 3′-end (primer 2, B and C) were incubated with 0.03 DNA polymerase units of p53/pol-prim in the presence of the radioactively labeled correct dNTP [dTTP to elongate primer 1 after the paired dCMP (A); dCTP to exchange the mispaired dGMP of primer 2 with the correct dCMP (B)] or incorrect dNTP [dATP to exchange the mispaired dGMP of primer 2 into the incorrect dAMP (C)]. For comparison, 16mer primers radioactively labeled at their 5′-end were loaded in lanes M (primer 1) and M* (primer 2). The sequence of the template and the hybridized primers 1 and 2 are presented on the left.

DISCUSSION

The finding that p53 possesses a 3′–5′-exonuclease activity and that p53 forms a complex with the replicative pol-prim, which does not contain an intrinsic 3′–5′-exonuclease activity (29–38), leads to the question of whether p53 can act as a proofreader for this particular DNA polymerase and whether it increases the fidelity of DNA replication. Using independently purified pol-prim and p53 from cultured cells, it was suggested that p53 might increase the accuracy of DNA polymerase α by excising potential DNA polymerase errors at the 3′-end (30). The observation that p53 interacts directly with pol-prim (29) was further extended and it was determined that p53 binds physically to pol-prim in vivo and that these proteins could be immunoprecipitated. Moreover, both enzymes p53 and pol-prim co-purified as a complex which was stable enough to sustain gel filtration (data not shown). Hereby, only a complex containing recombinant p53 and pol-prim possessed 3′–5′-exonuclease as well as DNA polymerase activity, and could remove the 3′-mispaired base of a primer, and elongated such a substrate whereas neither of these proteins alone could elongate such a primer.

As pol-prim is an essential replicative enzyme complex which lacks a proofreader, it has been hypothesized that errors made by this DNA polymerase can be corrected by the exonucleases of either DNA polymerases δ or ɛ (62). Another suggestion is that the primer-removing apparatus consisting of DNA helicase/endonuclease DNA2 and endonuclease FEN-1 removes that part of a nascent Okazaki fragment which has been synthesized by DNA polymerase α (63–65). Although the failure to find an efficient proofreading function for the p53/pol-prim complex would be in accord with the earlier observation that transgenic mice with an impaired p53 function do not exhibit a measurable increase in the frequency of point mutations in vivo (66,67), a more recent study reported a 2.3-fold higher mutation frequency in thymic lymphomas of p53–/– mice relative to that of the p53+/+ control (68). Furthermore, inactivation of p53 by the HPV16 E6 protein increased mutagenesis at the HPTR locus in human cells (69).

After purification of p53/pol-prim, the functional cooperation of DNA polymerase and exonuclease activity was studied using various in vitro assays. p53-containing pol-prim excised preferentially a 3′-mispaired primer end over a paired one. Furthermore, during this reaction the enzyme complex removed a terminal mispaired nucleotide present on a primer-template complex and replaced it with a correctly paired nucleotide. When all four dNTP substrates were in the reaction mixture, p53-containing pol-prim but not pol-prim alone was able to elongate a mispaired primer end (data not shown). p53 in the absence of pol-prim subunits generated only exonuclease products in this reaction mixture. It is the first time that the elongation of a mispaired substrate has been shown in addition to the excision of the mispaired nucleotide.

This reaction is p53 specific because in the presence of the exonuclease-inhibiting antibodies Pab421 and PAb240 p53/pol-prim could not extend a 3′ mispair similarly as reported before (30,32). These findings give further credit to the view that the exonuclease associated with p53 is an intrinsic part of this protein. In contrast with wild-type p53, a pol-prim complex containing the hot spot mutant p53R248H did not display exonuclease activity and did not elongate a mispaired 3′-end (70). These results confirm and extend the already published observations that the exonuclease activity of p53 degrades duplex DNA with a mismatch more rapidly than matched DNA and that p53 suppresses the incorporation of unpaired nucleotides by pol-prim (30,32–34). These in vitro findings supported the view that p53 might fulfill a proofreading function for pol-prim.

The p53/pol-prim complex can exchange a 3′ end mispaired with a correctly paired end but the efficiency of subsequent elongation of this newly generated paired end is poor compared with the elongation of the correctly paired end substrate. One obvious speculative explanation for this observation is that the exonuclease and the DNA polymerase activity of this purified enzyme complex do not fully cooperate. This suggestion is underlined by the finding that p53/pol-prim elongates a paired 3′-end as efficiently as does pol-prim although the exonuclease efficiently removes a mispaired nucleotide. The elongation activity of the complex appears hindered in elongating the newly formed paired primer end. These exchange and subsequent elongation processes are probably too slow in this purified system to be of benefit as a proofreading activity. It might be that additional factors are required to regulate the p53 exonuclease and to couple its activity to that of the pol-prim to optimize the cooperation of the enzymes. One of these factors could be RPA whose titration in these kinds of assays shows a 1.5–2-fold increased exchange reaction of mispaired nucleotides. Also, other factors or modifications are conceivable such as phosphorylation and acetylation (71,72). This view is in agreement with various in vivo experiments which show that wild-type p53 but not mutant p53 can increase fidelity of genomic DNA synthesis in living cells.

Acknowledgments

ACKNOWLEDGEMENTS

We thank F. Grosse and R. W. P. Smith for the critical reading of the manuscript and discussions, M. Willitzer for the synthesis and purification of oligonucleotides and J. Fuchs for technical assistance. This study was supported by grants from the Deutsche Krebshilfe/Dr Mildred Scheel Stiftung 10-1127-Gr1; 10-1560-Gr2 and the Deutsche Forschungsgemeinschaft. The IMB is a Gottfried-Wilhelm-Leibniz-Institut and financially supported by the federal government and by the state of Thüringen.

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