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: implications for fidelity and polymerase switching of translesion synthesis
1 Graduate School of Frontier Biosciences, Osaka University, and CREST, Japan Science and Technology Corporation, 1–3 Yamada-oka, Suita, Osaka 565–0871, Japan
2 Graduate School of Pharmaceutical Sciences, Osaka University, 1–6 Yamada-oka, Suita, Osaka 565–0871, Japan
3 Graduate School of Engineering Science, Osaka University, 1–3 Machikaneyama, Toyonaka, Osaka 560–8531, Japan
4 Cellular Physiology Laboratory, RIKEN Discovery Research Institute, Wako-shi, Saitama 351–0198, Japan
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Abstract |
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(pol ), which catalyses efficient translesion synthesis past cis-syn cyclobutane thymine dimers (TT dimers) and other lesions. The fidelity of DNA synthesis by pol on undamaged templates is extremely low, suggesting that pol activity must be restricted to damaged sites on DNA. Little is known, however, about how the activity of pol is targeted and restricted to damaged DNA. Here we show that pol binds template/primer DNAs regardless of the presence of TT dimers. Rather, enhanced binding to template/primer DNAs containing TT dimers is only observed when the 3'-end of the primer is an adenosine residue situated opposite the lesion. When two nucleotides have been incorporated into the primer beyond the TT dimer position, the pol -template/primer DNA complex is destabilized, allowing DNA synthesis by DNA polymerases 
or 
to resume. Our study provides mechanistic explanations for polymerase switching at TT dimer sites. |
Introduction |
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, and 
in eukaryotic cells, synthesize DNA with high fidelity, and they cannot replicate past DNA damage (Echols & Goodman 1991). In contrast, Y-family DNA polymerases, such as pol IV and pol V in E. coli, and pol , 
, 
, and Rev1 in mammalian cells, synthesize DNA with low fidelity, and they tend to bypass damage on template DNA (Boudsocq et al. 2002; Friedberg et al. 2002; Goodman 2002; Kunkel et al. 2003). Among this latter group of polymerases, pol is unique in its outstanding ability to catalyse efficient replication across the cis-syn cyclobutane thymine dimer (TT dimer), a lesion induced by UV, in an accurate manner (Masutani et al. 1999a, 2000; Johnson et al. 2000; Washington et al. 2000; Kusumoto et al. 2002).
Loss of human pol leads to the XP-V syndrome (Johnson et al. 1999; Masutani et al. 1999b). XP-V is an autosomal recessive human disease associated with an extreme sensitivity to sunlight and a high incidence of skin cancer. Cultured cells from patients are sensitive to UV irradiation and exhibit abnormal DNA replication after UV irradiation that is characterized by an enhanced mutation rate with altered spectrum (Lehmann et al. 1975; Maher et al. 1976; Wang et al. 1991, 1993; Misra & Vos 1993; Waters et al. 1993; Raha et al. 1996). Extracts from XP-V cells cannot support translesion synthesis (TLS) past defined TT dimers. The addition of pol is able to correct both the TLS defect of these cell extracts and the UV sensitivities of XP-V cells (Masutani et al. 1999a, 1999b; Yamada et al. 2000; Stary et al. 2003). These results indicate that the XPV gene product, human pol ? principally contributes to accurate TLS on templates containing TT dimers. However, the fidelity of pol is extremely low on undamaged templates (Johnson et al. 2000; Matsuda et al. 2000; Kusumoto et al. 2002), suggesting that cells have mechanisms to specifically load pol on to the site of a lesion and to remove it after TLS, when it is replaced with DNA polymerases that more accurately carry out chromosomal DNA replication. Recent findings strongly suggest that protein–protein interactions, especially between pol and PCNA (Haracska et al. 2001a, 2001b; Kannouche et al. 2001, 2004), are important for the TLS by pol . Theoretically, in addition to the protein–protein interactions, protein–DNA interactions may be features of mechanisms that control DNA polymerase switching. Here, we spotlight the DNA binding property of pol by itself and demonstrate an intriguing nature that may restrict its synthetic activity to the minimum required to bypass a DNA lesion. The results may explain how the switch from pol to a processive and high fidelity DNA polymerase such as pol takes place.
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Results |
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preferentially binds template/primer DNA substrates
To analyse the DNA binding activities of human pol , we employed electrophoretic mobility shift assay (EMSA) using the set of defined DNA substrates summarized in Table 1. The truncated form of human pol consisting of the amino-terminal 511 residues (pol (1–511)), which has a good solubility compared to the full-length protein, was used for the analyses. The protein corresponds to the originally identified form of an XP-V correcting protein which corrects TLS defects of XP-V cell extracts in cell-free systems by incorporating adenines opposite TT dimers (Masutani et al. 1999a, 1999b), indicating that the protein contains essential elements for in vitro functions of pol as a TLS polymerase. Although recent findings indicate that the carboxy-terminus of pol contains important elements for in vivo functions of human pol , i.e. a nuclear localization signal and residues interacting with PCNA, pol and Rev1 (Kannouche et al. 2001, 2003; Ohashi et al. 2004), the pol (1–511) should be valuable for the examination of the basic property of pol .
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to bind single-stranded, double-stranded, and template/primer DNAs with or without a TT dimer (Fig. 1). The dimer is situated on the 30-mer template strand just after the 3' end of the 16-mer primer in a template/primer DNA substrate, T30dimer/P16. When various 32P-labelled DNAs were incubated with pol , the resulting protein-DNA complexes could be observed as shifted bands after non-denaturing gel electrophoreses. These bands migrated more slowly when anti-pol antibodies were added to the reaction mixtures, showing that the shifted bands resulted from a specific interaction of pol with template DNAs (data not shown). As pol synthesizes DNA in the presence of magnesium ions and deoxyribonucleotides even on ice, we performed EMSA under the condition where they are absent to avoid making the results difficult to be interpreted. As far as we did, however, additions of these components rarely affected the EMSA profiles of the pol (data not shown).
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preferentially binds template/primer DNA substrates, weakly binds single-stranded DNA, and poorly binds double-stranded DNA. No significant difference was observed between undamaged and TT dimer-containing substrates for a given pair (compare T30 with T30dimer, T30/P16 with T30dimer/P16, and T30/P30 with T30dimer/P30), suggesting that by itself, pol is unlikely to recognize DNA damage.
The presence of the TT dimer does not inhibit the binding of DNA polymerase to the template/primer DNA substrate
Next, we examined the DNA binding activities of pol to template/primer DNAs that could be produced during TLS. Figure 2 shows the efficiency of pol binding to the 49-mer templates annealed with the 27-mer or 28-mer primer. In the T49dimer/P27 substrate, the 3' end of the 27-mer primer is positioned just before the TT dimer in the 49-mer template, which is similar to what was observed for the T30dimer/P16 substrate, as described above. In the case of the T49dimer/P28A, 3'-end nucleotide of the primer, A, was situated opposite the 3'T of the TT dimer. As shown in Fig. 2A,B, pol bound the T49dimer/P27 substrate as well as it bound the undamaged version, confirming the result with the 30-mer templates shown in Fig. 1. The KD values caluculated as concentrations of pol required for half-maximal substrate binding were 0.69 nM, 0.69 nM, 0.70 nM, and 0.54 nM for T49/P27, T49/P28A, T49dimer/P27, and T49dimer/P28A, respectively, indicating that the presence of the TT dimer does not inhibit the binding of pol to the template/primer DNA substrate.
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, the Klenow fragment bound template/primer substrates better than single-stranded and double-stranded DNAs, when these substrates did not contain TT dimers (data not shown). When the Klenow fragment was incubated with the T49dimer/P27, however, lesser amounts of the protein-DNA complexes were observed than for complexes containing the undamaged substrate. In the case of the T49dimer/P28A, the extent of binding of the Klenow fragment to the DNA was obviously much lower than for the other substrates, indicating that the presence of the TT dimer opposite the 3'-end nucleotide of the primer significantly reduces the affinity of the Klenow fragment for the DNA, in contrast to what was observed for pol . No damage bypass DNA polymerase activity was detected for the Klenow fragment with these template/primer DNAs containing TT dimers, while pol bypassed these lesions efficiently (data not shown). Thus, the ability of pol to bind a template/primer DNA regardless of the presence of the TT dimer may make it unique as a TLS polymerase.
The DNA polymerase -template/primer DNA complex is stabilized by the presence of a 3'-end primer nucleotide opposite the TT dimer
To assess the stabilities of pol -DNA complexes, we performed a dissociation assay in which pol was first incubated with various 32P labelled DNA probes, followed by the addition of cold competitor DNA. An excess of unlabelled T49dimer/P28A was added as a competitor to preformed complexes containing pol and labelled T49/P27, T49/P28A, T49dimer/P27, or T49dimer/P28A template/primer probes. After incubation, samples were loaded onto a non-denaturing gel to separate the competition-resistant pol -DNA complexes from the free DNA probes. Upon the addition of increasing amounts of competitor, pol dissociated from the probes, as shown in Fig. 3A,B. Interestingly, the pol -T49dimer/P28A complex was more resistant to competition than were complexes containing the T49dimer/P27 or undamaged DNA templates (the T49/P27 and T49/P28A substrates), suggesting that pol binds the TLS intermediate more stably than other substrates. In other words, once pol starts TLS by incorporating dAMP opposite a TT dimer, the pol -template/primer DNA complex is stabilized. This stabilization could be advantageous by allowing pol to continue TLS processively without dissociating from the templates containing TT dimers.
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-template/primer DNA complex formation
We next examined the influence of the nucleotide opposite the TT dimer on the stabilization of the pol -DNA complex by using template/primer DNA probes consisting of the TT dimer template and primers having different nucleotides at their 3' ends. Figure 4 shows the result of a dissociation assay with the 49 mer templates containing the TT dimer annealed with 28-mer primers which have different nucleotides at their 3' ends (T49dimer/P28A, T49dimer/P28C, T49dimer/P28G and T49dimer/P28T). Pol was incubated with each of these labelled substrates, and various amounts of an excess of unlabelled T49dimer/P28A were added as a competitor. Pol dissociated more readily from the T49dimer/P28C, T49dimer/P28G and T49dimer/P28T substrates upon the addition of the competitor than from the T49dimer/P28A substrate (Fig. 4A,B). These results suggest that pol continues to bind the DNA only when it incorporates a correct nucleotide ‘A’ opposite the TT dimer, and that it easily dissociates from the complex if it incorporates an incorrect nucleotide.
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-template/primer DNA complex is destabilized after the primer is extended two nucleotides beyond the TT dimer
We further examined the affinities of pol for template/primer DNAs that may be generated during or after TLS. Figure 5 shows the results of dissociation assays with 49-mer templates annealed with 29-mer, 30-mer, and 31-mer primers. In the T49dimer/P29 substrate, the 3' end nucleotide of the primer is positioned in front of the 5'T of the TT dimer. In the T49dimer/P30 and T49dimer/P31 substrates, the 3' nucleotides of the primers are positioned one and two nucleotide(s) beyond the TT dimer, respectively. Pol was incubated with these labelled substrates, and various amounts of an excess of unlabelled T49dimer/P28A were added as a competitor to the preformed complexes. As shown in Fig. 5A,B, pol -DNA complexes with the T49dimer/P29 and T49dimer/P30 substrates were relatively resistant to competition, as was the pol -T49dimer/P28A complex (compare with Fig. 3), but the complex with the T49dimer/P31 substrate was not. When undamaged substrates (T49/P29, T49/P30 or T49/P31) were used, complex stabilization was not observed (Fig. 5C,D). These results suggest that once pol starts the translesion reaction by incorporating a first dAMP opposite the 3'T of the TT dimer, the pol -DNA complex is stabilized, which allows pol to incorporate the next nucleotide. The stability of the pol -DNA complex is maintained during the incorporation of the second dAMP opposite the 5'T of the TT dimer and of the next nucleotide beyond the lesion. After the incorporation of the fourth nucleotide (the second nucleotide beyond the lesion), the pol -DNA complex is no longer stable, and pol dissociates from the DNA.
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to permit the resumption of DNA synthesis by replicative DNA polymerases and
After translesion synthesis mediated by pol , replicative DNA polymerases are thought to resume DNA replication. To determine the number of nucleotides pol must add before it is replaced with a replicative polymerase, we examined the abilities of the replicative DNA polymerases pol and pol to extend P27, P28A, P29, P30, and P31 primers annealed to the T49dimer template (the T49dimer/P27, T49dimer/P28A, T49dimer/P29, T49dimer/P30 or T49dimer/P31 substrates). As shown in Fig. 6A, pol could not extend primers whose 3' end was situated just before the TT dimer (the T49dimer/P27 substrate) or opposite either T of the TT dimer (the T49dimer/P28A and T49dimer/P29 substrates), consistent with our previous observations (Masutani et al. 2000). Pol could barely extend a primer whose 3' end was situated just after the TT dimer (the T49dimer/P30 substrate), but it was fully capable of extending a primer whose 3' end was situated two nucleotides after the TT dimer (the T49dimer/P31 substrate) (Fig. 6A). The synthesis reaction with the T49dimer/P31 substrate was as efficient as that with the undamaged template/primer substrate (Fig. 6C). Thus, DNA synthesis by pol up to two nucleotides beyond the TT dimer is necessary and sufficient to allow pol to resume DNA replication. The main terminal product synthesized by pol with the template/primer substrate was one nucleotide shorter than expected, given the length of the template. This product length is independent of the presence of the TT dimer, because the same profile was observed when the undamaged T49/P31 substrate was extended by pol (Fig. 6C). It is likely that pol has difficulty in synthesizing DNA up to the end of this 49-mer template, perhaps due to its 5'-terminal sequence content.
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, pol did not give elongated products with the T49dimer/P27, T49dimer/P28A, and T49dimer/P29 template/primer DNAs, regardless of the presence of PCNA (Fig. 6B). As pol has a 3' 
5' exonuclease activity, degraded products were also detected. The major product of the exonucleolytic reaction with all three substrates was the labled 27-mer, suggesting that the degradation mediated by pol was arrested just before the lesion. Indeed, synthesis mediated by pol as well as by pol stopped just before the lesion when a shorter primer annealed to the 49-mer template was used (data not shown). When pol was incubated with the T49dimer/P30 or T49dimer/P31 substrates, in which the primer extends beyond the TT dimer of the template by one or two correct base pairs, degradation and extension products were both observed, and production of the latter was increased by the addition of PCNA in both cases. Importantly, the 31-mer primer was elongated more efficiently than was the 30-mer primer. These results suggest that it is favourable for pol to resume DNA synthesis after the incorporation of two nucleotides beyond the lesion by pol . Taken together with the observation that pol readily dissociates from the T49dimer/P31 substrate (Fig. 5), we suggest that pol incorporates two nucleotides opposite the TT dimer and two more nucleotides thereafter, and the replicative DNA polymerase then replaces pol to continue DNA synthesis. |
Discussion |
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results in an UV-sensitive and cancer-prone syndrome, XP–V, indicating that it is involved in a damage tolerance mechanism that protects the genome from UV-induced abnormalities, including mutations. Pol , however, has an intrinsic mutagenic property, suggesting that there are cellular regulatory mechanisms that limit its activity and/or increase its fidelity. Here we demonstrate that pol activity is intrinsically limited to a minimum.
TT dimers block DNA synthesis by replicative DNA polymerases such as pol and pol . These DNA polymerases cannot incorporate nucleotides opposite TT dimers and stop DNA synthesis just before lesions. The translesion DNA polymerase pol is therefore expected to restart DNA synthesis by incorporating a nucleotide opposite the damaged base. As shown in Fig. 6, pol and pol may efficiently resume DNA synthesis from a primer DNA that has two extra nucleotides beyond the position of the TT dimer, suggesting that overall, pol catalyses the addition of four nucleotides, two nucleotides opposite the two thymidine residues of the TT dimer, and two nucleotides beyond the lesions. The observed DNA binding properties of pol meet these requirements perfectly. Thus, our findings have implications for the behaviour of pol after it binds a damaged template. Based on our results, we propose a model for DNA polymerase switching during TLS across TT dimers (Fig. 7). (1) Pol replaces a replicative DNA polymerase stalled just before the TT dimer. (2) Pol incorporates a correct nucleotide, ‘A’, opposite the 3'T of the dimer, resulting in its stable association with the TLS intermediate. (3) The pol -DNA complex remains stable after the incorporation of nucleotides opposite the 5'T of the dimer and the next nucleotide beyond the lesion, suggesting that pol can polymerize four nucleotides on the TT dimer template without dissociating from it. (4) After the incorporation of the second nucleotide beyond the lesion, pol no longer stably binds the template/primer substrate, resulting in its dissociation from the DNA. (5) A template/primer substrate whose primer end has two extra nucleotides beyond the lesion can be efficiently elongated by replicative polymerases. Thus, the biochemical properties of pol minimize the association of the enzyme with the template DNA. On the other hand (6), if pol incorporates C, G or T opposite the 3'T of the dimer, the pol -DNA complex is destabilized, and (7) pol tends to dissociate from the DNA. (8) It is thought that misincorporated nucleotides are proofread by 3' 5' exonucleases (Bebenek et al. 2001). Pol could then be reloaded on to the edited product and elongation could resume. Thus, the DNA binding properties of pol may contribute to the fidelity of synthesis. Pre-steady-state kinetic studies have suggested that like classical DNA polymerases, pol undergoes an induced-fit conformational change when catalysing nucleotide incorporation (Washington et al. 2001). Unlike replicative polymerases, however, pol poorly discriminates between correct and incorrect nucleotides by this nucleotide incorporation mechanism. Thus, TLS mediated by pol does not include selectivity with respect to incorporate nucleotides. Considered in the context of our findings, the conformational change that may occur after the correct nucleotides are incorporated opposite the TT dimer is very important for relatively accurate TLS by pol . Structural analyses of Y-family polymerases, including yeast pol and archaeal DinB homologues, reveal that they have large active sites that accomodate the TT dimer (Ling et al. 2001; Trincao et al. 2001; Zhou et al. 2001). A model based on recent structural studies of the archaeal Dpo4 complexed with TT dimer-containing DNA and incoming nucleotides proposes that pol changes conformation during TLS across the TT dimer (Ling et al. 2003). Our data can provide a biochemical basis for this model.
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, human cells have three other Y family TLS polymerases: pol , pol and Rev1. Moreover, pol 
, a member of the B family of polymerases, has TLS activity against various DNA lesions (Nelson et al. 1996; Guo et al. 2001; Johnson et al. 2003; Xie et al. 2003). How do cells select a suitable DNA polymerase at sites of lesions? The stabilization of the pol -DNA complex was not observed with templates containing (6–4) photoproducts, which are poorly bypassed by pol (data not shown), suggesting that the DNA binding abilities of TLS polymerases are closely related to their synthetic activities in the presence of specific lesions. There may be other mechanisms, however, by which pol and/or other DNA polymerases are loaded on to substrates containing lesions, as we were unable to detect a selective recognition ability of pol for damaged DNA. Pol , pol and pol are reported to interact with PCNA (Haracska et al. 2001a, 2002), which is known to stimulate the processivity of pol . It has also been reported that modifications of PCNA with ubiquitin and SUMO are mediated by the RAD6 pathway, and that these alterations regulate damage tolerance mechanisms including TLS (Hoege et al. 2002; Stelter & Ulrich 2003). Thus, PCNA could be a key factor in the selection of the DNA polymerases during switching. Here we examined for the truncated form of human pol and found some intrinsic natures of the protein, but it is obviously necessary and interesting to get on the analyses with the full-length pol in the co-ordinate actions with PCNA.
As described above, pol and pol were found to interact with each other (Kannouche et al. 2003). More recently, mammalian Rev1 was found to interact with other Y-family polymerases, pol , pol , and pol in a yeast two-hybrid assay and by co-immunoprecipitation experiments (Guo et al. 2003; Ohashi et al. 2004). These protein–protein interactions may be involved in regulating polymerase switching at sites bearing lesions that cause polymerase to stall. Our present working hypothesis is as follows; Upon stalling of the replication apparatus at the site of a DNA lesion such as a TT dimer, Rad6p and Rad18p modify PCNA by mono-ubiquitination, resulting in the activation of translesion synthesis by TLS polymerases such as pol (Hoege et al. 2002; Stelter & Ulrich 2003; Kannouche et al. 2004). In some cases, other TLS polymerases may be recruited to stalled replication sites by polymerase–polymerase interactions depending on the type of the DNA lesion (Guo et al. 2003; Ohashi et al. 2004). The actual polymerase switching event occurs at the lesion site through the association of pol with TT dimer-containing template/primer DNA. After incorporation of two nucleotides opposite the TT dimer and two more nucleotides beyond the TT dimer, pol dissociates from the DNA, allowing replicative polymerases such as pol and pol to resume DNA synthesis (present study). The results of our recent experiments using primer extension reactions with TT dimer-containing template/primers and purified pol are consistent with this model (McCulloch et al. 2004). Reconstitution of the whole reaction in a cell-free system is expected to further enhance our understanding of mechanism of translesion DNA synthesis.
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Experimental procedures |
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Plasmid pBS.XPV, which carries human POLH cDNA cloned into the EcoRI site of pBluescript II KS+, was digested with NotI, and the resulting 3.5 kb cDNA fragment was isolated (Masutani et al. 1999b) and cloned into the NotI site of pRcCMV to generate pRcCMV-XPV. The translational initiation and termination sites of pol in the plasmid pRcCMV-XPV were converted to NdeI and XhoI sites with the oligonucleotides 5'-GTC CAG TAG CCA TAT GTC AAG GTA AC-3' and 5'-GCC TGA GGG CAC TCG AGA TGT GTT AAT GG-3', respectively, using a site-directed mutagenesis system, Mutan-K (Takara Shuzo), essentially according to the method described by Kunkel et al. (1987). The resulting plasmid was termed pRcCMV-XPV-NdeI-XhoI. To obtain a truncated POLH cDNA fragment encoding pol (1–511)-His, an additional XhoI site was introduced into the pRcCMV-XPV-NdeI-XhoI using the oligonucleotide 5'-GTA ATG AGG GCT TGG ACT CGA GAT TGC TCA TGG GAG CCT G-3' and the Mutan-K system, and the resulting plasmid was digested with NdeI and XhoI. The truncation fragment was cloned into the NdeI and XhoI sites of pET21b (Novagen) to add His tag sequences. The resulting plasmid, pET21b-XPV(1–511)His, was digested with Bpu1102I and the recessed 3'-ends were made blunt with T4 DNA polymerase. The DNA was further digested with XbaI to obtain the XPV(1–511)His fragment. A baculovirus expression vector, pFast BacI (Life Technologies Inc.), was digested with XhoI, blunt-ended with T4 DNA polymerase, and further digested with XbaI. The XPV(1–511)His cDNA was ligated to the digested vector, thus generating pFB I-XPV(1–511)His. Recombinant baculovirus was generated with this construct using the Bac-to-bac expression system (Life Technologies Inc.).
Proteins
Recombinant human pol (1–511) tagged with hexa-histidine at the carboxy terminus was expressed in Sf9 insect cells using the baculovirus expression system and purified to near homogeneity by sequential column chromatography on Hitrap Q (Amersham Pharmacia), Ni-NTA agarose (Qiagen) and Mono S (Amersham Pharmacia) columns, as previously described (Masutani et al. 2000). The protein concentration was determined using Amido Black with BSA as a control (Schaffner & Weissmann 1973). DNA polymerases and were purified from mouse FM3A and HeLa cells, as described (Lee et al. 1989; Eki et al. 1991). PCNA was expressed in E. coli BL21 (DE3) and purified as described (Fien & Stillman 1992).
Electrophoretic mobility shift assay
The following methods were adapted from published protocols (Masuda et al. 1998). Oligomers containing the CPD were synthesized as described (Murata et al. 1990). Standard binding reactions (10 µL) were carried out on ice for 15 min in mixtures including 25 mM potassium phosphate buffer (pH 7.4), 0.2 mg/mL BSA, 5 mM dithiothreitol, 2.5% glycerol, 15 mM KCl, 20 mM NaCl, 5'-32P-labelled DNA (5 fmol), poly dI-dC (1 ng) and the indicated amount of recombinant human pol (1–511)His. For dissociation assays, binding reactions with labelled DNAs were carried out on ice for 30 min, unlabelled DNAs were then added, and the mixtures were incubated on ice for 15 min. The mixtures were directly loaded on 4% non-denaturing polyacrylamide gels (acrylamide:bis-acrylamide, 79 : 1) and electrophoresed at 8 V/cm at 2 °C in TAE buffer (6 mM Tris-HCl (pH 7.5), 5 mM sodium acetate, 0.1 mM EDTA). The gels were dried and exposed to X-ray film at –80 °C. The percentage of the probe DNA bound to pol (1–511)His was determined using the BAS2500 bioimaging analyser (Fujifilm).
DNA polymerase assay
Standard 10 µL reactions contained 25 mM potassium phosphate buffer (pH 7.4), 5 mM MgCl2, 0.1 M dNTPs, 0.2 mg/mL BSA, 5 mM dithiothreitol, 2.5% glycerol, 15 mM KCl, 20 mM NaCl, 5'-32P-labelled DNA (320 fmol), DNA polymerase and PCNA. Reactions were performed at 37 °C for 15 min and terminated by adding 10 µL formamide followed by boiling. Products were electrophoresed on 20% polyacrylamide-7 M urea gels and autoradiographed.
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Acknowledgements |
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Footnotes |
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aPresent address: National Institute on Ageing, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
* Correspondence: E-mail: fhanaoka{at}fbs.osaka-u.ac.jp |
References |
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Received: 14 July 2004
Accepted: 31 August 2004
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lanes 1–6; •lanes 7–12; x lanes 13–18; 
lanes 19–24. The mean values and standard errors were calculated from two independent experiments.
