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Genes to Cells (2006) 11, 3-11. doi:10.1111/j.1365-2443.2005.00918.x
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Visualization of the interaction between archaeal DNA polymerase and uracil-containing DNA by atomic force microscopy

Yasuo Asami1,a, Masahiro Murakami2, Masatomi Shimizu1,3, Francesca M. Pisani4, Isamu Hayata2 and Takehiko Nohmi1,*

1 Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501 Japan
2 Radiation Hazards Research Group, Research Center of Radiation Safety, National Institute of Radiological Sciences 4-9-1, Anagawa, Inage-ku, Chiba-shi, Chiba, 263-8555 Japan
3 Department of Food and Nutrition, Aobagakuen Junior College, 3-12-9 Setagaya, Setagaya-ku, Tokyo, 154-0017 Japan
4 Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino, 111. 80131-Napoli, Italy


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B-family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double-stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil-containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Deamination of exocyclic amino groups of DNA bases such as deamination of the amino group of cytosine poses a common genotoxic risk in all organisms (Lindahl 1993; Pearl 2000). The hydrolytic deamination of cytosine leads to the formation of uracil in DNA, and G:U base pairs result in G:C to A:T transitions in a half of the progeny if not repaired before replication. Since the rate of the hydrolytic reaction is greatly accelerated at high temperatures (Lindahl & Nyberg 1974), hyperthermophilic organisms, which live in habitats at more than 80 °C, are supposed to be exposed to massive DNA damages by the deamination. However, the spontaneous mutation rate in hyperthermophilic archaea Sulfolobus acidocaldarius is reported to be similar to that of Escherichia coli (Grogan et al. 2001). Thus, hyperthermophilic archaea appear to possess mechanisms to protect a stability of the genomic DNA from the mutagenic threat of deaminated bases generated at high temperatures.

Interestingly, B-family DNA polymerases from hyperthermophilic archaea such as Sulfolobus solfataricus DNA polymerase B1 (Pol B1) or Pyrococcus furiosus DNA polymerase (Pfu) recognize the presence of uracil in DNA and tightly bind to uracil-containing oligonucleotides (Lasken et al. 1996; Greagg et al. 1999). Pol B1 is likely to play an important role in DNA replication in S. solfataricus because the activity is highly stimulated by PCNA-like and RFC-like factors in in vitro (De Felice et al. 1999). Pol B1 and Pfu stall DNA polymerization three to four base pairs (bps) before template uracil. The recognition and stalling mechanisms by the polymerases appear to contribute to the genome integrity of hyperthermophilic archaea because they may prevent the misincorporation of adenine opposite the template uracil. More surprisingly, a recent study suggests that the enzymes also bind to the deamination product of adenine, i.e. hypoxanthine, in a template DNA strand and stall DNA synthesis upstream of the lesion (Gruz et al. 2003). Hypoxanthine can pair with cytosine, thereby inducing A:T to G:C transitions if not repaired (Lindahl 1993). Thus, the recognition mechanisms of deaminated bases by archaeal B family DNA polymerases may play more important roles in maintaining the genome stability than previously thought. The recognition mechanism seems unique to archaeal B family DNA polymerases because viral B family DNA polymerases such as T4 DNA polymerase or DNA polymerases from hyperthermophilic eubacteria such as Thermus aquaticus (Taq) do not stall DNA synthesis when the template DNA has uracil or hypoxanthine (Greagg et al. 1999; Gruz et al. 2003). There are no specific reports on the recognition of uracil in DNA by eukaryotic B-family DNA polymerases as far as we know. Structural analysis for uracil recognition by archaeal B family DNA polymerases indicates a pocket in the N-terminal domains interacting with a template strand is responsible for the discrimination of uracil from normal DNA bases (Fogg et al. 2002).

Since Pol B1 is abundantly expressed in the cell, i.e. 1500 molecules per S. solfataricus cell, it may bind to the deaminated bases in the chromosome DNA even without DNA synthesis. In fact, it binds to uracil- or hypoxanthine-containing oligonucleotides without primers or dNTPs necessary for DNA synthesis (Gruz et al. 2003). If such a binding occurs in vivo, it may interfere with normal repair of the deaminated bases by DNA glycosylases (Sartori et al. 2002). Thus, there appear to be mechanisms that prevent non-productive binding of Pol B1 to the deaminated bases in non-replicating chromosomes. For further insights into the binding mechanisms of archaeal B family DNA polymerases to the deaminated bases in DNA, we compared the binding efficiencies of Pol B1 to uracil in DNA with or without DNA synthesis. For this purpose, we employed atomic force microscopy, which is suitable for the analysis of the behavior of individual molecules, as well as gel mobility shift assays (Engel & Muller 2000; Murakami et al. 2000). The results indicated the binding of Pol B1 to uracil in DNA is greatly accelerated by DNA synthesis and suggested that Pol B1 is targeted to the deaminated bases in replicating DNA. In addition, analyses with atomic force microscope (AFM) suggested that Pol B1 binds to uracil-containing DNA as a monomer, which directly supports the "read-ahead" mechanism where single molecules of DNA polymerase bind to template uracil and halt DNA replication (Greagg et al. 1999).


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Binding of Pol B1 to uracil-containing DNA is accelerated by DNA synthesis

To examine the binding of Pol B1 to uracil in single-stranded DNA (ssDNA), control or U100 DNA, which contains uracil instead of thymine in DNA, was incubated with various amounts of Pol B1, and the migration of DNA bands was analyzed by gel mobility shift assays (Fig. 1). A clear band shift was observed when U100 DNA was incubated with Pol B1. A smear band appeared in the upper part of the gel while the original DNA bands remained in the lower part. In these experiments, primers and dNTPs were omitted from the reaction mixtures. Thus, it seems that Pol B1 can bind uracil-containing ssDNA even without DNA synthesis. This is consistent with our previous results that the Kd values of Pol B1 to primed DNA, ssDNA without uracil and ssDNA with uracil are 81, 55 and 4 nM, respectively (Gruz et al. 2003). It should be noted, however, that the binding was not efficient: it needed a large amount of Pol B1, i.e. 400 pmol, and no band shift was observed with lower amounts of the protein. The binding appears specific to uracil-containing DNA because no clear band shift was observed in control DNA.



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  		Figure 1  Binding of Pol B1 with template uracil without DNA synthesis. The reaction mixture (20 µL) contained Tris/KCl/MgCl2, ssDNA (control or U100 DNA, 2 pmol) and Pol B1 (2, 20, 200 or 400 pmol), and the mixture was incubated for 15 min at 55 °C. The products were analyzed by 1% agarose gel electrophoresis, followed by Southern hybridization. The bands were visualized with ChemiDoc.—no Pol B1 was added in the reaction mixture.

 
Next, we examined the inhibitory effects of template uracil on DNA synthesis with U100 DNA (Fig. 2). DNA synthesis by Pol B1 was severely inhibited by the presence of uracil in template DNA. ssDNA containing uracil (U100 DNA) was hardly converted to double-stranded DNA (dsDNA), while control ssDNA was almost completely converted to dsDNA within 10 min. Most of ssDNA bands of U100 diminished during 10- or 30-min incubation without formation of dsDNA. Since Pol B1 possesses an exonuclease proofreading activity (Pisani & Rossi 1994), uracil-containing ssDNA might be partially digested by the exonuclease during the incubation. Like DNA synthesis by Pol B1, DNA synthesis by another archaeal B-family DNA polymerase, i.e. Pfu exo–, was severely inhibited by the presence of uracil in template DNA. In this case, however, ssDNA bands were observable even after 30-min incubation. The lack of exonuclease activity in Pfu exo– DNA polymerase might account for the persistence of the ssDNA bands. In contrast to the archaeal enzymes, DNA synthesis by hyperthermophilic eubacterial DNA polymerase, i.e. Taq, was not inhibited by the presence of template uracil.



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  		Figure 2  Template uracil inhibits DNA synthesis by Pol B1 and Pfu exo– but not by Taq DNA polymerase. The reaction mixture (20 µL) contained Tris/KCl/MgCl2, F1 primer (10 pmol), four dNTPs (4 nmol each), ssDNA (control or U100 DNA, 0.1 pmol) and Pol B1 (20 pmol). Taq DNA polymerase (1 unit) and Pfu exo– DNA polymerase (2.5 units) were used as controls. The reaction was carried out for 10 or 30 min at 55 °C and was terminated by the addition of EDTA. The products were analyzed by Southern hybridization and visualized with ChemiDoc. Taq, Taq DNA polymerase; Pfu exo–, Pfu DNA polymerase exo–; Pol B1, DNA polymerase B1.

 
Since template uracil strongly inhibited DNA synthesis by Pol B1, the binding of Pol B1 during DNA synthesis might be stronger than that of Pol B1 without DNA synthesis. To examine the possibility, we analyzed the binding during DNA synthesis by the gel shift assay (Fig. 3). When Pol B1 and U100 DNA were incubated with primer and dNTPs, a smear band appeared on the gel. The band intensity was much stronger with U100 DNA than with control DNA. When we exposed the gel for shorter period of time, it became evident that substantial amounts of U100 DNA disappeared during the incubation (data not shown). It should be emphasized that the amount of Pol B1 (20 pmol, Fig. 3) required for binding with U100 DNA was 1/20 of that required for the binding to U100 DNA without primer and dNTPs in the reaction mixture (400 pmol, Fig. 1). These results suggest that the binding efficiency of Pol B1 with template uracil is substantially enhanced by DNA synthesis.



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  		Figure 3  Specific binding of Pol B1 with template uracil during DNA synthesis. The reaction mixture (20 µL) contained Tris/KCl/MgCl2, F1 primer (10 pmol), four dNTPs (4 nmol), ssDNA (control or U100 DNA, 2 pmol) and Pol B1 (20 pmol). The mixtures were incubated for 10 min at 55 °C, and the reactions were terminated by the addition of EDTA. The products were analyzed by Southern hybridization and visualized with ChemiDoc.

 
Pol B1 binds to uracil-containing DNA as a monomer

Atomic force microscopy is a powerful and convenient tool to visually analyze the behavior of individual DNA and protein molecules (Argaman et al. 1997; Murakami et al. 2001). The AFM method was employed to directly characterize the features of ssDNA, dsDNA and Pol B1 at the single molecular level (Fig. 4A). In the image analysis, ssDNA and dsDNA appeared as spherical and linear forms, respectively (Forms I and II). Pol B1 appeared as smaller spherical forms (Form III). The observed length of dsDNA, i.e. 500 nm, was consistent with the calculated length of 1.4 kb DNA (0.32 nm x 1400 = 448 nm). The reaction mixtures containing Pol B1, ssDNA (Control or U100 DNA), primers and dNTPs were subjected to AFM analysis before and after incubation for 15 min at 55 °C (Fig. 4B). There were many Form I (ssDNA) and Form III (Pol B1) molecules in the mixtures before the incubation. After the incubation, however, Form II (dsDNA) became apparent in the reaction mixtures containing control DNA. In contrast, Form II (dsDNA) was rare in the mixtures containing U100 DNA, and other forms such as those where Pol B1 appeared to bind to DNA were noted (Form IV, Fig. 4A,C). The sizes of the bound molecules (50-70 nm) were similar to those of monomeric forms of Pol B1 (Fig. 4A,D).



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  		Figure 4  AFM images of DNA, Pol B1 and intermediates where Pol B1 binds with uracil-containing DNA. (A) Classification of the images of ssDNA (Form I), dsDNA (Form II), Pol B1 (Form III) and other forms including the intermediates (Form IV). (B) AFM images of the products of DNA synthesis with Pol B1 plus control DNA or U100 DNA. The reaction mixtures before and after incubation were analyzed by AFM. (C) Typical images of ssDNA, dsDNA, Pol B1 and the intermediates. (D) Possible explanation for the AFM image of the intermediate Form IV.

 
To analyze the dsDNA formation more quantitatively, individual molecules on the mica were counted, and the percentage of each form among total molecules was calculated (Table 1). It is evident that Form II (dsDNA) was generated, concomitant with the reduction of the number of Form I (ssDNA) in the reaction mixture containing control DNA, Pol B1, primers and dNTPs. The number of Form II (dsDNA) increased to a quarter of the total number of molecules (22/88 total molecules) while that of Form I (ssDNA) decreased to one third of it (30/88 total molecules). In contrast, Form II (dsDNA) accounted for only 5% of the total number of molecules (9/201 total molecules) and Form I (ssDNA) represented more than 60% (123/201 total molecules) in reaction mixtures containing U100 DNA, Pol B1, primers and dNTPs. The differences in the distribution of each form between two reaction products were statistically significant (P < 0.0001 by {chi}2 test). These results confirm the inhibitory effects of template uracil on DNA synthesis by Pol B1. Form IV (DNA plus Pol B1) accounted for about 41% (36/88 total molecules) and 34% (69/201 total molecules), respectively, of total numbers of molecules in the two reaction products. These values are significantly larger than the percentages of Form IV observed in the samples of Control (13% = 43/343), U100 DNA (9% = 9/104), Control DNA plus Pol B1 (14% = 15/104) and U100 DNA plus Pol B1 (18% = 29/158), suggesting that DNA synthesis accelerates the formation of Form IV.


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  		Table 1 Distribution of three forms of AFM images in various reaction products
 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Uracil is one of the most ubiquitously occurring mutagenic damages in DNA (Lindahl 1993). It is generated by deamination of cytosine in DNA as well as by incorporation of dUTP into DNA by DNA polymerases (Vassylyev & Morikawa 1996). Since the deamination of cytosine results in G:U base pairs, and the uracil in template DNA directs DNA polymerases to incorporate adenine opposite it, the hydrolytic reaction can convert original G:C base pairs to A:T base pairs in a half of the progeny. To exclude uracil from DNA, organisms possess various uracil-DNA glycosylases, which specifically excise uracil in DNA (Pearl 2000), and dUTPases, which hydrolyze dUTP to dUMP plus PPi to maintain low levels of dUTP in the cellular nucleotide pool (Hogrefe et al. 2002). In addition, hyperthermophilic archaeal B family DNA polymerases such as Pol B1 or Pfu possess unique mechanisms that recognize uracil in template DNA and inhibit the progress of DNA synthesis upstream from the lesion (Greagg et al. 1999). Given the genotoxicity of uracil in DNA, the recognition mechanism by the polymerases appears to contribute to the maintenance of genome integrity of the organisms living in the habitats at high temperatures.

To better understand the binding mechanisms, we examined the binding modes of Pol B1 to uracil in DNA by gel mobility shift assays and atomic force microscopy. We used the same ssDNA substrates, i.e. control and U100 DNA, whose molecular sizes are 1.4 kb, throughout the analyses. These DNAs were chosen because the linear form of dsDNA (Form II) can be clearly visible with AFM and is distinct from Form III (Pol B1). In addition, the ssDNA is efficiently converted to dsDNA by Pol B1 within 10 min at 55 °C (Fig. 2). The efficient conversion from ssDNA to dsDNA suggests that the ssDNA does not form substantial intramolecular base interactions at 55 °C, thereby allowing the progress of DNA polymerase on the template strand. Although Pol B1 itself could specifically bind to ssDNA containing uracil, the molar ratio between ssDNA containing uracil (U100 DNA) and Pol B1 required for the band shift was 1 : 200 in the absence of DNA synthesis (Fig. 1). The presence of uracil in the ssDNA strongly inhibited DNA syntheses by Pol B1 and Pfu exo– but not by Taq DNA polymerases (Fig. 2). The inhibitory effect was also clearly observed when the formation of dsDNA (Form II) in reaction products containing U100 DNA in the presence of Pol B1, primers and dNTPs was analyzed by atomic force microscopy (Table 1). The percentage of dsDNA was significantly lower in the reaction mixture containing U100 DNA (5%) than in that containing control DNA (25%).

Since the presence of uracil in DNA strongly inhibits the DNA synthesis by DNA pol B1, we postulated DNA pol B1 might bind to uracil in DNA in the progress of DNA synthesis more efficiently compared to during random diffusion in solution. Interestingly, the binding efficiency of Pol B1 to uracil was greatly enhanced by initiating DNA synthesis (Fig. 3). The molar ratio between U100 DNA and Pol B1 was 1 : 10 when the reaction mixtures contained primers and dNTPs necessary for DNA synthesis. Thus, we suggested that Pol B1 binds more efficiently to uracil when it proceeds along with template DNA for DNA synthesis rather than during random collision with DNA in solution (Fig. 5). This conclusion is consistent with the result that the percentage of Form IV (DNA plus DNA pol B1) in the mixture containing DNA pol B1 and U100 DNA was significantly higher in the presence of DNA synthesis (34%) than in the absence (18%) (Table 1). As described in the Introduction, the expression level of Pol B1 is 1500 molecules per cell, which is noticeably higher than the ones reported for E. coli DNA polymerases (Gruz et al. 2003). Because of the preferential binding of Pol B1 to uracil during DNA synthesis, we suggest that the majority of Pol B1 molecules are engaged in DNA replication and recognize uracil during DNA synthesis rather than directly binding to uracil in the non-replicating chromosome as lesion-specific binding proteins. Since Pol B1 is less sensitive in sensing uracil in the template strand than Pfu exo- (Gruz et al. 2003), which binds tightly to uracil in template/primer DNA (Shuttleworth et al. 2004), we suggest that Pol B1 might continue DNA synthesis beyond template uracil to some extent and randomly halt replication before template uracil along with template DNA (Fig. 5). In fact, we observed Pol B1 bound to uracil-containing DNA at various positions in the template DNA. It should be noted that Pol B1 possesses 3' to 5' exonuclease activity (Pisani & Rossi 1994). Thus, the primer strand may be digested at least in part when Pol B1 encounters uracil in template DNA during DNA synthesis in vivo.



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  		Figure 5  Schematic model of the mechanism of recognition of uracil in DNA by Pol B1. (A) Random diffusion-mediated recognition. Pol B1 randomly diffuses in solution and binds to uracil in DNA. (B) DNA replication-mediated recognition. Pol B1 slides on template DNA for elongation of primer and binds to uracil in DNA.

 
Since archaeal DNA polymerases bind to uracil-containing ssDNA, it is possible that one polymerase molecule actively extending the primer is blocked by a second polymerase bound to uracil in the template strand (Greagg et al. 1999). This "blocking" model seems plausible in particular for the long-range primer extension, and contrasts with the "read-ahead" model where single molecules of DNA polymerases directly bind to uracil in the template DNA and stop replication. To distinguish the two possibilities, we used AFM and suggested that Pol B1 bound to uracil-containing DNA as a monomeric form (Fig. 4). This finding directly supports the "read-ahead" model as a mechanism by which archaeal DNA polymerases halt copying DNA before template uracil. Since Form IV (DNA plus DNA pol B1) was observed even in "Control" or "U 100 DNA" samples where no Pol B1 was added, some of Form IV observed were not necessarily true replication intermediates where DNA polymerases bound to DNA. However, Form IV was more frequently generated when DNA replication was initiated (Table 1). Thus, we suggest that a significant portion of Form IV observed in the samples of "Control + Pol B1 + dNTPs + Primer" and "U100 DNA + Pol B1 + dNTPs +Primer" include the true replication intermediates. The sizes of the molecules bound to control DNA and U100 DNA in the presence of dNTPs and primers were indistinguishable. Hence, we suggested that monomeric forms of Pol B1 bind to uracil in template DNA. However, more extensive quantification of the size of many of the blobs at the end of the dsDNA in different conditions is needed for the final conclusion.

Both Pol B1 and Pfu exo– bind not only to template uracil but also to template hypoxanthine, a deamination product of adenine (Gruz et al. 2003). Pfu exo– stalls three to four bps before hypoxanthine as well as uracil in the template strand, and Pol B1 displays a similar stalling behavior. Hypoxanthine pairs with cytosine during DNA replication and induces A:T to G:C transitions if not repaired. Thus, this stalling behavior seems to suppress the incorporation of cytosine opposite template hypoxanthine, thereby reducing the mutagenic potential of hypoxanthine. Stalling Pol B1 at template uracil or hypoxanthine may generate ss gap DNA region downstream of the lesions. Recombination that seals the gap with DNA sequence of sister chromatid or translesion DNA synthesis with Y-family DNA polymerase may contribute to the gap filling (Fig. 6). In fact, Sulfolobus solfataricus possesses a homolog of Rad51, i.e. RadA, a recombination protein, and a homolog of E. coli DNA pol IV, i.e. Sso DNA pol Y1 or Dpo4, a Y-family DNA polymerase(Seitz et al. 1998; Gruz et al. 2001; Ohmori et al. 2001; She et al. 2001). In addition, it has uracil DNA glycosylase to excise uracil in DNA (She et al. 2001). Thus, it is important to investigate how these proteins are involved in the subsequent steps leading to repair of deaminated bases in archaeal DNA.



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  		Figure 6  Possible mechanisms by which daughter strand gaps generated by stalling of Pol B1 at template uracil (or hypoxanthine) are sealed by homologous recombination and/or translesion bypass by Pol Y1. Cytosine in DNA (a) is deaminated by heat, thereby generating G:U mismatch (b). When the strand containing uracil is copied by Pol B1 before the uracil is removed by repair enzymes, Pol B1 may stop before uracil (c). ssDNA region downstream of the uracil can be sealed by homologous recombination with DNA sequence from sister chromatid (d). Uracil DNA glycosylase may excise the uracil (e) and provides a chance to regenerate normal G:C bp (a). However, if Pol B1 is switched to Pol Y1, the Y-family DNA polymerase may bypass uracil by incorporating adenine opposite template uracil (f). Then, another polymerase switch occurs and Pol B1 extends the primer strand containing adenine opposite uracil, thereby generating a mutagenic mispair in DNA (g).

 

   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Preparation of ssDNAs with or without uracil

TaKaRa Taq DNA polymerase, Pfu DNA polymerase exo–, and FPLC-grade dNTPs were purchased from TaKaRa (Shiga, Japan), Stratagene (La Jolla, CA, USA) and Amersham Biosciences, respectively. Pol B1 was purified as previously described (Pisani & Rossi 1994). About 1.4 kb DNA was amplified by PCR using plasmid pUC118 DNA (3.2 kb), F1-biotin primer (5' biotin-GGGAGAAAGGCGGACAGGTA-3', 20 pmol), R2 primer (5'-GGCTGGCTTAACTATGCGGC-3', 20 pmol), Taq DNA polymerase (1 unit) and four dNTPs (4 nmol each) in a total volume of 20 µL. Taq DNA polymerase almost equally incorporates dUTP and dTTP opposite template adenine (Lasken et al. 1996). Advantage was taken of this property and 1.4 kb DNA containing uracil was prepared by the same PCR conditions except for the presence of dUTP (4 nmol) instead of dTTP. The former and latter amplified DNAs were named "control DNA" and "U100 DNA," respectively. After the amplification, excess primers were digested with ssDNA specific exonuclease, i.e. ExoSAP-IT (Amersham Biosciences). The proteins were inactivated by heat and proteinase K treatments, and removed by phenol/chloroform/isoamylalcohol extraction. The DNAs were purified by ethanol precipitation, and re-suspended in TE buffer. After removal of low-molecular-weight contaminants by filtration with Microcon YM100 (Millipore, Bedford, MA, USA), ssDNAs were prepared by heating (100 °C x 5 min) followed by rapid cooling on ice. The resulting ssDNAs were used for the analysis with AFM. ssDNAs for gel shift assays were prepared with magnetic streptavidin bead according to the manufacturer's protocol (MAGNOTEX-SA, TaKaRa, Japan). Briefly, the filtrated DNAs having biotin were mixed with MAGNOTEX-SA, and ssDNAs without biotin were obtained in the supernatant after treatments with alkaline solution. The incorporation of uracil into DNA was confirmed by treating the DNAs with uracil DNA glycosylase (Gibco BRL, Gaithersburg, MD, USA), which converts uracil into abasic sites in DNA (Krokan et al. 2002). U100 DNA, but not control DNA, became a poor substrate for PCR amplification with Taq because of the generation of abasic sites.

Gel mobility shift assay

Interactions between Pol B1 and ssDNA with or without uracil were examined under various conditions, the details of which are described in the legends of Figs 1–4. Briefly, the reaction mixture (20 µL) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl and 1.5 mM MgCl2 (Tris/KCl/MgCl2). In addition, it contained F1 primer (5'-GGGAGAAAGGCGGACAGGTA-3'), four dNTPs, Pol B1 and ssDNA. When the interactions were analyzed without DNA synthesis, F1 primer and dNTPs were omitted from the mixture. The reaction was carried out at 55 °C and terminated by the addition of 1/10 volume of 40 mM ethylenediaminetetraacetic acid (EDTA) solution. The reaction products were separated by electrophoresis with 1% agarose gel, and transferred to nylon membrane (Hybond-N+, Amersham Biosciences) for Southern hybridization analysis. Probe DNA (0.7 kb) was prepared by PCR in a reaction mixture containing pUC118, F1 primer and R1 primer (5'-GGCCTCTTCGCTATTACGCC-3'). R1 primer anneals a DNA sequence close to the multiple-cloning site of pUC118, while F1 and R2 primers anneal DNA sequences each 0.7 kb apart from the cloning site in opposite directions. The amplified DNA was fluorescently labeled and used for the hybridization with ECL Direct Nucleic Acid Labeling and Detection System (Amersham Biosciences). The hybridized DNA was quantified using a chemiluminescent detection system, i.e. ChemiDoc (Bio-Rad, Richmond, CA, USA).

AFM analysis

The reaction mixture (40 µL) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 20 pmol each of R2 and F1 primers, 8 nmol each of four dNTPs, 1 pmol Pol B1 and 2 pmol of ssDNA (control or U100 DNA). The reaction was carried out for 15 min at 55 °C, and the samples were directly deposited on to freshly cleaved and discharged mica, followed by a rinse with distilled water and blown dry with dry nitrogen. The plates were placed in a desiccator for 1 h before analysis. The samples were visualized with AFM (Model SPI 3800 N, Seiko Instruments Inc., Japan) in dynamic force mode (DFM) operation with a 20 µm scanner (Murakami et al. 2000, 2001). The cantilevers for the DFM-AFM (Micro Cantilever, Type SI-DF40, Seiko Instruments Inc., Japan) were used for the analysis. Images were collected at room temperature. Scan frequencies were typically 2.0 Hz, and all images contain 512 x 512 data points.


   Acknowledgements
 
Part of this study was financially supported by the Budget for Nuclear Research of the Ministry of Education, Culture, Sports, Science and Technology, Japan, based on the screening and counseling by the Atomic Energy Commission. This work was also supported by Grants-in-aid for Cancer Research from the Ministry of Health, Labour and Welfare, Japan, and for International Collaborative Research from the Japan Health Science Foundation.


   Footnotes
 
Communicated by: Fumio Hanaoka

aPresent address: Department of Molecular Biotechnology, Hiroshima University, 1-3-1 Kagamiyama, Hiroshima 739-8530, Japan Back

* Correspondence: E-mail: nohmi{at}nihs.go.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Argaman, M., Golan, R., Thomson, N.H. & Hansma, H.G. (1997) Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope. Nucleic Acids Res. 25, 4379–4384.[Abstract/Free Full Text]

De Felice, M., Sensen, C.W., Charlebois, R.L., Rossi, M. & Pisani, F.M. (1999) Two DNA polymerase sliding clamps from the thermophilic archaeon Sulfolobus solfataricus. J. Mol. Biol. 291, 47–57.[CrossRef][Medline]

Engel, A. & Muller, D.J. (2000) Observing single biomolecules at work with the atomic force microscope. Nature Struct. Biol. 7, 715–718.[CrossRef][Medline]

Fogg, M.J., Pearl, L.H. & Connolly, B.A. (2002) Structural basis for uracil recognition by archaeal family B DNA polymerases. Nature Struct. Biol. 9, 922–927.[CrossRef][Medline]

Greagg, M.A., Fogg, M.J., Panayotou, G., Evans, S.J., Connolly, B.A. & Pearl, L.H. (1999) A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil. Proc. Natl. Acad. Sci. USA 96, 9045–9050.[Abstract/Free Full Text]

Grogan, D.W., Carver, G.T. & Drake, J.W. (2001) Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Proc. Natl. Acad. Sci. USA 98, 7928–7933.[Abstract/Free Full Text]

Gruz, P., Pisani, F.M., Shimizu, M., et al. (2001) Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C. J.Biol. Chem. 276, 47394–47401.[Abstract/Free Full Text]

Gruz, P., Shimizu, M., Pisani, F.M., De Felice, M., Kanke, Y. & Nohmi, T. (2003) Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus. Nucleic Acids Res. 31, 4024–4030.[Abstract/Free Full Text]

Hogrefe, H.H., Hansen, C.J., Scott, B.R. & Nielson, K.B. (2002) Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation. Proc. Natl. Acad. Sci. USA 99, 596–601.[Abstract/Free Full Text]

Krokan, H.E., Drablos, F. & Slupphaug, G. (2002) Uracil in DNA—occurrence, consequences and repair. Oncogene 21, 8935–8948.[CrossRef][Medline]

Lasken, R.S., Schuster, D.M. & Rashtchian, A. (1996) Archaebacterial DNA polymerases tightly bind uracil-containing DNA. J.Biol. Chem. 271, 17692–17696.[Abstract/Free Full Text]

Lindahl, T. (1993) Instability and decay of the primary structure of DNA. Nature 362, 709–715.[CrossRef][Medline]

Lindahl, T. & Nyberg, B. (1974) Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry 13, 3405–3410.[CrossRef][Medline]

Murakami, M., Hirokawa, H. & Hayata, I. (2000) Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies. J. Biochem. Biophys. Meth 44, 31–40.[Medline]

Murakami, M., Minamihisamatsu, M., Sato, K. & Hayata, I. (2001) Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy. J. Biochem. Biophys. Meth 48, 293–301.[Medline]

Ohmori, H., Friedberg, E.C., Fuchs, R.P., et al. (2001) The Y-family of DNA polymerases. Mol. Cell 8, 7–8.[CrossRef][Medline]

Pearl, L.H. (2000) Structure and function in the uracil-DNA glycosylase superfamily. Mutat. Res. 460, 165–181.[Medline]

Pisani, F.M. & Rossi, M. (1994) Evidence that an archaeal alpha-like DNA polymerase has a modular organization of its associated catalytic activities. J.Biol. Chem. 269, 7887–7892.[Abstract/Free Full Text]

Sartori, A.A., Fitz-Gibbon, S., Yang, H., Miller, J.H. & Jiricny, J. (2002) A novel uracil-DNA glycosylase with broad substrate specificity and an unusual active site. EMBO J. 21, 3182–3191.[CrossRef][Medline]

Seitz, E.M., Brockman, J.P., Sandler, S.J., Clark, A.J. & Kowalczykowski, S.C. (1998) RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange. Genes Dev. 12, 1248–1253.[Abstract/Free Full Text]

She, Q., Singh, R.K., Confalonieri, F., et al. (2001) The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. USA 98, 7835–7840.[Abstract/Free Full Text]

Shuttleworth, G., Fogg, M.J., Kurpiewski, M.R., Jen-Jacobson, L. & Connolly, B.A. (2004) Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea. J.Mol. Biol. 337, 621–634.[Medline]

Vassylyev, D.G. & Morikawa, K. (1996) Precluding uracil from DNA. Structure 4, 1381–1385.[Medline]

Received: 10 July 2005
Accepted: 3 October 2005





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