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J Biol Chem, Vol. 274, Issue 36, 25599-25607, September 3, 1999

Mode Analysis of a Fatty Acid Molecule Binding to the N-terminal 8-kDa Domain of DNA Polymerase
A 1:1 COMPLEX AND BINDING SURFACE^*

Yoshiyuki Mizushina, Tadayasu Ohkubo^§, Takayasu Date^¶, Toyofumi Yamaguchi, Mineo Saneyoshi, Fumio Sugawara, and Kengo Sakaguchi^**

From the  Department of Applied Biological Science, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, the ^§ Japan Advanced Institute of Science and Technology, 15 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan, the ^¶ Department of Biochemistry, Kanazawa Medical University, Uchinada, Kahoku-gun, Ishikawa 920-0293, Japan, and the  Department of Biological Science, Teikyo University of Science and Technology, Yamanashi 409-0193, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase . At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C₁₈-linoleic acid (LA) or C₂₄-nervonic acid (NA). In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48-51, and 79-87) and residues adjacent to an -type loop connecting helix-1 and helix-2 of the same face. No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain. The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K_D) of 1.02 or 2.64 mM, respectively.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We reported previously that long-chain fatty acids strongly inhibited the activities of mammalian DNA polymerase  (pol )¹ and DNA polymerase  (pol ) in vitro and plant DNA polymerases, albeit less potently, but that at the concentrations used, the fatty acids hardly influenced the activities of prokaryotic DNA polymerases or other DNA metabolic enzymes such as DNase I (1, 2). The most potent inhibitors were fatty acids, which have the following characteristics: hydrocarbon chain containing 18 or more carbons, a free carboxyl end, and the cis-configuration is preferred to the trans-configuration. Fatty acids in the trans-configuration have a much weaker inhibitory effect on pol , and those in which the carboxyl end is chemically modified can lose the inhibitory effect on both pol  and pol . The mode of inhibition by longer chain fatty acids showed the same characteristics, except that the minimum inhibitory doses of these longer chain fatty acids were much lower (2, 3). Lineweaver-Burk plots of the fatty acids indicated that both the substrate (i.e. deoxynucleotide)-binding and the template DNA-binding sites of pol  were nonantagonistically inhibited by the fatty acids, but they were effective as antagonists against the sites of pol . For pol , fatty acids acted by competing with not only the substrate but also the template-primer DNA. In screening inhibitors of eukaryotic DNA polymerase, we also found several natural compounds which inhibited pol  in the same manner as fatty acids (4-10).

Pol  is the smallest known DNA polymerase in animal cells with a molecular mass of 39 kDa, and its structure is highly conserved among mammals (11). This protein has a modular two-domain structure, with apparent flexibility within a protease-sensitive region between residues 82 and 86, which separates the two domains. Treatment with trypsin yields an N-terminal domain fragment (8 kDa), which retains binding affinity for single-stranded DNA (ssDNA), and a C-terminal domain fragment (31 kDa) with reduced DNA polymerase activity (12, 13). We reported previously the mode of biochemical inhibition by fatty acids using two of the pol  fragments that were proteolytically separated (3). The fatty acids were found to bind to the 8-kDa DNA-binding domain fragment and to suppress binding to the template-primer DNA. A 10,000-fold higher level of fatty acid was required for binding to the 31-kDa catalytic domain or to inhibit the DNA polymerase activity, suggesting that it directly disturbs the template-primer incorporation into the template-primer-binding domain and indirectly competes with the substrate on its binding site in the catalytic domain (3). The binding between the enzymes and fatty acids can be released by detergents without any permanent damage to the structure (2, 3). These results suggested that the binding is physiologically specific and has some roles in vivo: for example, to maintain the enzymes in the inactive state on the internal surface of the membranes. In this study, we analyzed the structural interactions of C₁₈ and C₂₄ fatty acids with pol , especially the N-terminal 8-kDa domain, in cross-linking studies using a 5'-end-labeled photoprobe (dT₁₄D) instead of template-primer DNA and the binding surface of the 8-kDa domain in contact with the fatty acids by NMR.

The crystal and NMR structures of pol  and the N-terminal 8-kDa domain of pol  have been determined recently by Wilson and his co-workers (14-22). Based on the information available from their studies of crystal and NMR analyses of pol  and the 8-kDa domain (14-22), we compared the interaction interface for ssDNA template with that for the fatty acids. The study of the relationship between fatty acids and pol  may reveal why C₁₆ or shorter fatty acids cannot inhibit the polymerase activity (2), why the minimum inhibitory doses of longer chain fatty acids are much lower than those of shorter chain species, although the biochemical mode of inhibition is the same, and how the fatty acids bind to the N-terminal 8-kDa domain. These studies may help to further clarify the structure and function of pol  and subsequently may allow us to speculate on the in vivo role of DNA polymerase inhibition by fatty acids.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Sample Preparation-- The N-terminal 8-kDa fragment of rat DNA pol  (residues 2-87) was overexpressed in Escherichia coli strain BL21 harboring the expression plasmid "Lys-87" constructed in our laboratory. Overproduction of the N-terminal 8-kDa domain and the purification procedure have principally been described in our previous report (23). For ¹⁵N-correlated NMR experiments, the N-terminal 8-kDa domain was expressed from BL21/Lys-87 grown on minimal medium containing ¹⁵NH₄Cl as the sole nitrogen source (24). In preparing the NMR sample, the purified N-terminal domain was concentrated using a Centricon-3 (Amicon) and exchanged into 5 mM potassium phosphate buffer (pH 7.0) and 20% D₂O. Two samples for NMR experiments contained 1.25 mM ¹⁵N-labeled N-terminal 8-kDa domain after addition of fatty acids (linoleic acid (LA) and nervonic acid (NA)). The fatty acids (12.5 mM each) were dissolved in Me₂SO-d⁶. Fragments of 8 and 31 kDa of pol  were prepared and purified as described previously (3).

Photoaffinity Cross-linking of Proteolytic Fragments of Pol  with Photolabile dT₁₄D-- The reaction mixture (9.4 µl) was comprised of 50 mM Tris-HCl (pH 8.8), 1 mM dithiothreitol, 100 mM KCl, 0.5 mM MnCl₂, 0.085 µM ³²P-5'-end-labeled photoprobes dT₁₄D (i.e. an oligothymidylate 15-mer analogue containing a photolabile 2'-deoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl)styryl]uridylate residue at the 3' terminus, 0.8 pmol/reaction mixture) (25), and 20% Me₂SO with various concentrations of fatty acids. The 8-kDa domain fragment (0.14 µg, 18 pmol) and 31-kDa domain (0.55 µg, 18 pmol) or 8-kDa domain fragment (0.14 µg, 18 pmol) were added in the reaction mixture. Incubation was carried out for 5 min at 25 °C. After chilling at 0 °C, the reaction mixture was irradiated with a near-UV lamp as described (25) for 10 min in an ice bath. The product was analyzed by SDS-PAGE (12.5% separation gel) (26) and detected by autoradiography.

Circular Dichroism Spectroscopy-- For CD analysis, 6.25 µM (50 µg/ml) purified 8-kDa domain fragment of pol  and 62.5 µM fatty acid mixture (molecular ratio, enzyme:inhibitor = 1:10) were dissolved in 5 mM potassium phosphate buffer (pH 7.0) containing 5% methanol. The chromatograms for the mixtures were compared with that of the 8-kDa domain alone. The concentrations of the proteins were determined by UV adsorption at 280 nm (₂₈₀ = 5440 M¹ cm¹). CD measurements were performed on a Jasco J-720 spectropolarimeter in a 1-cm cell at 25 °C. The CD spectra were collected from 260 to 200 nm at a resolution of 1 nm using up to eight scans. The per residue molar ellipticity (degree·cm² dmol¹) was calculated from the concentration for the 87-residue polypeptide.

NMR Experiments-- NMR spectra were measured at 750 MHz on a Varian Unity-Plus 750 spectrometer. ¹H-¹⁵N HMQC spectra were recorded at a temperature of 30 °C. Each spectrum size was 1024 complex points in the t2 dimension and 96 complex points in t1. The data were zero filled in both dimensions, and a shifted sine-bell was applied as a window function for resolution enhancement. A total of 32 scans per FID was accumulated, leading to a measuring time of 90 min per HMQC spectrum.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The in vitro relationship between mammalian DNA polymerases and fatty acids has been investigated (2, 3). As described in the Introduction, longer chain fatty acids (over 18 carbons) strongly inhibited DNA polymerase activities in vitro. Fatty acids in which they are of the trans-configuration have much weaker inhibitory effects on especially pol , and the fatty acids in which the carboxyl end is chemically modified can lose the inhibitory effect. In this study, we analyzed the structure of pol  and its relationship to the long-chain fatty acids in more detail. Lineweaver-Burk plots of fatty acids indicated that both the substrate-binding and the template DNA-binding sites of pol  were antagonistically inhibited by fatty acids (2, 3). We tested fatty acids from C₁₈ to the longest commercially available, C₂₄, previously (3). Among the fatty acids examined, the strongest inhibitor was a C₂₄ fatty acid, nervonic acid (NA), and the weakest was a C₁₈ fatty acid, linoleic acid (LA) (3). We therefore analyzed the mode of binding to pol  using the longest and the shortest fatty acids in the present study.

The effects of fatty acid were analyzed using proteolytic methods. Rat pol , which was also used in this study, can be divided into two domain fragments (8- and 31-kDa polypeptides) using controlled proteolysis (12, 13). The 8-kDa domain is the DNA template-binding domain, and the 31-kDa domain is the catalytic part involved in DNA polymerization. According to the methods described by Kumar et al. (12), we purified both of these fragments by fast protein liquid chromatography Superose 12 (lanes 3 and 4 in Fig. 4, Ref. 3) and used them in this experiment. We also suggested previously that the fatty acids bind to pol  and the 8-kDa domain fragment, but not to the 31-kDa domain fragment (3). Both NA and LA appear to interact with the enzyme or the 8-kDa domain fragment in the same way, but the longer chain binds to the domain fragment more tightly and inhibits DNA polymerase activity much more strongly.

Analysis of the Binding between Fatty Acids and Fragments of 8 and 31 kDa of Pol  by Using Cross-linking-- The labeling abilities of the synthesized ³²P 5'-end-labeled photoprobes (dT₁₄D) were investigated using the N-terminal 8-kDa and C-terminal 31-kDa domain fragments of the recombinant rat pol  (39 kDa). The ³²P 5'-end-labeled photoprobe (dT₁₄D) had a molecular mass of 5 kDa. dT₁₄D was mixed with 8- and 31-kDa fragments of pol  and irradiated with near-UV light as described under "Experimental Procedures," and then analyzed by gel mobility shift assay (Fig. 1). As shown in Fig. 1A, autoradiography of the radioactive products of the 8-kDa domain fragment resolved by SDS-PAGE showed a shift from the original 8-kDa to the 13 (8 + 5)-kDa position as the labeled protein complex (lane 1). Poly (dT), which is a DNA template, competed with the photoprobe for binding to the 8-kDa domain fragment (lanes 1-3 in Fig. 1A). The 8-kDa band observed in Fig. 1A may have been due to the absorption of very small amount of labeled photoprobe. A small amount of dT₁₄D could bind to the 31-kDa domain fragment and cause a shift to the 36 (31 + 5)-kDa position, although this band was faint (lane 1 in Fig. 1A). The SDS-PAGE characteristics of dT₁₄D and recombinant rat pol  (39 kDa) were shown previously (Fig. 6 of Ref. 25). When the mixture was not irradiated, no cross-linking of the proteins with dT₁₄D was observed (lane 4 in Fig. 1A). The 8-kDa domain fragment of pol  could be shifted with the cross-linked dT₁₄D, but the 31-kDa fragment, the catalytic domain without a DNA-binding site, could not, because this domain has no DNA binding capacity (lane 1 in Fig. 1A). Cross-linking of the fragments of pol  with photolabile dT₁₄D was inhibited by addition of the natural template, poly(dT) (lanes 1-3 in Fig. 1A), showing that the 8-kDa domain fragment has contained the template DNA-binding site.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Photoaffinity cross-linking of the N-terminal 8-kDa or the C-terminal 31-kDa domain fragment of rat DNA polymerase  with 32P-5'-end-labeled photolabile dT14D. The photoaffinity cross-linking experiments were carried out as described under "Experimental Procedures." The concentration of photolabile dT14D used was 0.8 pmol in each lane. A, the 8- and 31-kDa fragments were added at 18 pmol. The concentrations of poly(dT) are described in each panel. B and C, in the presence of 18 pmol of 8-kDa fragment and 0.8 pmol of dT14D, LA (B), or NA (C) was added at the concentration indicated in each panel.

LA and NA interfered with complex formation of the 8-kDa fragment and ssDNA template (Fig. 1,B and C). pT₁₄D at a concentration of 0.8 pmol was added to 18 pmol of the 8-kDa fragment along with various concentrations of LA (Fig. 1B) or NA (Fig. 1C). The I/E (inhibitor/enzyme) ratios in the presence of 0, 1.8, 3.6, and 7.2 µM fatty acids (LA is Fig. 1B, NA is Fig. 1C) were 0, 0.25, 0.5, and 1, respectively. NA interfered with binding of the DNA to the 8-kDa fragment, and at an I/E ratio of 1, the interference became nearly complete (Fig. 1C). We, therefore, concluded that one molecule of fatty acids competes with oligonucleotide, i.e. dT₁₄D, suggesting that a fatty acid molecule interferes with the binding of template DNA to one molecule of the 8-kDa domain fragment. Similar results were obtained using LA instead of NA (Fig. 1B). LA also interfered with DNA binding to the fragment, but the interference was not complete at an I/E ratio of 1. Thus, the C₂₄ fatty acid, NA, showed stronger interference than the C₁₈ fatty acid, LA, at the I/E ratio of 1. This may explain the observation that the inhibition of pol  activity by NA was 10-fold stronger than that by LA although the biochemical mode of inhibition was the same (2, 3).

To explain why the minimum inhibitory dose of the longer chain fatty acid was much lower than that of short chain species, the dissociation constants (K_D) between each of the fatty acids and the domain fragment were also analyzed as described in the later part of this report (Fig. 3). To investigate the binding mode including K_D in detail, NMR structures of the N-terminal 8-kDa domain with or without the fatty acids were determined.

CD Spectra of 8-kDa Domain and Mixture of 8-kDa Domain and Fatty Acids-- The CD spectra of complexes of the 8-kDa domain fragment and the fatty acids were very similar to the CD spectrum of the 8-kDa domain fragment alone (Fig. 2). The comparable maximal negative ellipticities at 208 and 220 nm indicated that the overall helical structure in the mixture of the 8-kDa domain, and the fatty acids were similar to that of the 8-kDa domain fragment alone (Fig. 2). The spectra of the protein-LA complex and the protein-NA complex were similar to each other, but the maximal negative ellipticity of 208 nm and the maximal positive ellipticity of 235-260 nm of the protein-NA complex were higher than those of the protein LA complex (Fig. 2). The unchanged ratio of the maximal negative ellipticity at 222 nm versus 208 nm in the mixture of the 8-kDa domain fragment and the fatty acids suggested no increase in helical structure in comparison with the 8-kDa domain fragment alone. On the basis of these results, we concluded that the fatty acids do not adversely affect the overall structure of the 8-kDa domain fragment.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   . CD spectra of the N-terminal 8-kDa domain of DNA polymerase  with the fatty acids. The CD spectra were collected as described under "Experimental Procedures." Black line, the 8-kDa domain only; dotted line, the mixture of the 8-kDa domain and LA; gray line, the mixture of the 8-kDa domain and NA.

Analysis of the Binding of Fatty Acids to the N-Terminal 8-kDa Domain by NMR-- The NMR structures of the N-terminal 8-kDa domain have recently been determined by Wilson, Mullen, and their co-workers (19). According to their results, the 8-kDa domain (residues 1-87) is formed by four -helices, packed as two antiparallel pairs. The pairs of -helices cross one another at 50° giving them a V-like shape. The 8-kDa domain contains a motif termed the "helix-hairpin-helix." The protein residues involved in template DNA binding have been identified by NMR using chemical shift changes (16). The helix-3-hairpin-helix-4 motif and residues in an adjacent -type loop connecting helix-1 and helix-2 form the ssDNA interaction surface (16). Furthermore, they also found that several mutants of the 8-kDa domain (F25W, K35A, K60A, and K68A) showed impaired template DNA binding activity (14). In a biochemical study, using the purified recombinant 8-kDa domain, photochemical cross-linking studies showed that residues Ser-30 and His-34 cross-linked to p(dT)₁₆ (27).

In studying the effects of fatty acid binding, the recombinant 8-kDa domain fragment was titrated with a 12.5 mM stock solution of LA or NA. Two-dimensional ¹H-¹⁵N HMQC spectra were recorded for the 8-kDa domain-fatty acid complex at fatty acid concentrations of 0.3125, 0.625, 0.9375, 1.25, 1.5625, 1.875, 2.1875, and 2.5 mM. The complex is in fast exchange on the time scale of NMR, permitting us to follow the chemical shift changes of the backbone NH and ¹⁵N signals of the 8-kDa domain upon complex formation by recording a series of ¹H-¹⁵N HMQC spectra of uniformly ¹⁵N-labeled 8-kDa domain in the presence of increasing amounts of fatty acids. Of the 79 amides in residues 5-86 of the 8-kDa domain, 75 amides were assigned in the fatty acid complex. The cross-peak for Leu-11 was sufficiently resolved during the titration to allow determination of the mole fraction of protein bound with fatty acids. The backbone amide of Leu-11 displays the longest chemical shift change upon complexation. The change in the chemical shift of the Leu-11 resonance is interpreted as resulting from the chemical shifts for the free (F) and the bound forms (B) of the Leu-11 resonance being averaged into a single resonance (av) (i.e. (F  B)k_off for the complex (28)). Fitting of the titration curve for the amide proton resonance of Leu-11 indicated that the 8-kDa domain binds to LA or NA as a 1:1 complex with a K_D of 1.02 or 2.64 mM, respectively (Fig. 3), indicating that the longer fatty acid could bind to the fragment more tightly. This probably explains why the minimum inhibitory dose of the longer chain fatty acid was much lower, although the biochemical mode of inhibition was the same. Since C₁₆ or shorter fatty acids are expected to have higher K_D values than the K_D of the C₁₈ fatty acid, this may also explain why the shorter chain fatty acids cannot inhibit polymerase activity. Fig. 4A shows the ¹H-¹⁵N HMQC spectrum of the 8-kDa domain alone. Fig. 4B shows the ¹H-¹⁵N HMQC spectrum of the 8-kDa domain (blue contours) overlaid on that of the 1:1 mixture of the 8-kDa domain and NA (red contours). Similarly, Fig. 4C depicts the superimposed spectra of the 1:1 mixture of the 8-kDa domain and NA (red contours) and the 1:1 mixture of the 8-kDa domain and LA (green contours). Chemical shift changes of 0.015 for ¹H and 0.1 for ¹⁵N were determined for Lys-5, Ala-6, Gln-8, Glu-9, Leu-11, Leu-22, Ala-23, Glu-26, Asn-28, Val-29, Ser-30, Ile-33, Lys-35, Asn-37, Tyr-39, His-51, Lys-52, Ile-73, Asp-74, Phe-76, Leu-77, Ala-78, Thr-79, Gly-80, Leu-82, and Lys-84 (Fig. 5). The data in Fig. 5 indicate the NH chemical shift differences in the presence of 1.25 mM LA or NA along the amino acid sequence of the 8-kDa domain in Fig. 4. The shifted cross-peaks for LA were the same as those for NA, except Leu-11 and Thr-79, suggesting that the mode of binding to the domain does not change between LA and NA.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Determination of KD for fatty acid binding to the N-terminal 8-kDa domain of DNA polymerase  (1.25 mM). Titration of LA or NA was performed to measure the chemical shift change at the nondegenerate Leu-11 amide proton in 1H-15N HMQC NMR. The KD values of LA and NA were 1.02 and 2.64 mM, respectively.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Expanded 1H-15N HMQC spectra of the 15N-labeled N-terminal 8-kDa domain-fatty acid complex. A, the N-terminal 8-kDa domain (1.25 mM) only. The values in the figure indicate the amino acid sequences of the 8-kDa domain peptide. B, the N-terminal 8-kDa domain (1.25 mM) in the absence (blue) or presence (red) of 1.25 mM NA. C, the N-terminal 8-kDa domain (1.25 mM) in the presence of 1.25 mM LA (green) or NA (red). In B and C, the major shifted cross-peaks of the amino acid residues are indicated as the amino acid sequence.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Chemical shift changes for the N-terminal 8-kDa domain of DNA polymerase  on complex formation with the fatty acids LA and NA. The chemical shift differences (the cross-peak shift values of the free domain minus those of the domain complex shown in Fig. 4) for the amide proton chemical shifts (A) or for the amide 15N chemical shifts (B) are shown in bars.

Mapping of the Fatty Acid Interaction Interface-- Fig. 6A shows the residues displaying chemical shift changes on binding to the fatty acids in the solution structure of the 8-kDa domain with or without fatty acids. NH chemical shift changes of 0.015-0.03 ppm and ¹⁵N chemical shift changes of 0.1-0.2 ppm (Lys-5, Leu-22, Ala-23, Asn-28, Asn-37, Tyr-39, Lys-52, Ile-73, Asp-74, Ala-78, Leu-82, and Lys-84) are shown in yellow. NH chemical shift changes of 0.03-0.06 ppm and ¹⁵N chemical shift changes of 0.2-0.4 ppm (Ala-6, Gln-8, Glu-9, Glu-26, Val-29, Ser-30, Ile-33, Phe-76, Leu-77, and Gly-80) are shown in orange. NH chemical shift changes of more than 0.06 ppm and ¹⁵N chemical shift changes of more than 0.4 ppm (Leu-11, Lys-35, His-51, and Thr-79) are shown in red. These exposed residues showing significant changes were the same between LA and NA (Figs. 4C and 5). In the presence of either LA or NA, the cross-peaks were shifted as follows: Lys-5, Ala-6, Gln-8, Glu-9, and Leu-11 were in the unstructured segment; Glu-26 was in helix-1, which is adjacent to the -type loop; Asn-28, Val-29, Ser-30, Ile-33, and Lys-35 were in the -type loop; Asn-37 and Tyr-39 were in helix-2, which is adjacent to the -type loop; His-51 and Lys-52 were in a turn; Ile-73, Asp-74, Phe-76, Leu-77, and Ala-78 were in helix-4, which is adjacent to the 48-55 turn and 79-87 unstructured linker segment; Thr-79, Gly-80, Leu-82, and Lys-84 were in the unstructured linker segment that connects to the 31-kDa catalytic domain in the full-length enzyme. These chemical shift changes can be explained in terms of the fatty acid contact and perturbation in the electrostatic charge distribution at the surface. Surface residues displaying chemical shift changes were predominantly, although not entirely, clustered on one side of the domain (Fig. 6A). Furthermore, the fatty acid-binding interface of the 8-kDa domain consists of two regions: one consisting of Leu-11 in the 1-13 unstructured segment, His-51 in the 45-55 turn, and Thr-79 in the 79-87 unstructured linker segment ("I" in Fig. 6A), while the other consists of an -type loop, including helix-1 and helix-2 ("II" in Fig. 6A).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Interaction between the fatty acid and the amino acid residues of the N-terminal 8-kDa domain of rat DNA polymerase  (Protien Data Base code 1BNO). The N-terminal domain consists of four helices, helix-1 (15-26), helix-2 (36-47), helix-3 (56-61), and helix-4 (69-78), tightly packed to form a hydrophobic core. The remainder of the domain consists of two turns (48-55 and 62-68), an -type loop (27-35), and extended structures (1-14 and 79-87). A, interactions between fatty acids and the 8-kDa domain. The amino acid residues of the major shifted cross-peaks from HMQC NMR experiments are indicated. 0.015-0.03 ppm of NH chemical shift changes and 0.1-0.2 ppm of 15N chemical shift changes are depicted in yellow. 0.03-0.06 ppm of NH chemical shift changes and 0.2-0.4 ppm of 15N chemical shift changes are indicated in orange. NH chemical shift changes at more than 0.06 ppm and 15N chemical shift changes at more than 0.4 ppm are indicated in red. B, interactions between ssDNA and the 8-kDa domain. These data were from the experiments reported by Wilson, Mullen, and their co-workers (14, 16, 19). The amino acid residues Phe-25, Lys-35, Lys-60, and Lys-68, which were shown to be necessary for ssDNA binding activity by site-directed mutagenesis (14), are indicated in blue. The amino acid residues of the major shifted cross-peaks from HMQC NMR experiments (16) are depicted in pink.

Fig. 6B shows the mapping in the solution structure of the 8-kDa domain with ssDNA. The data determined by Wilson, Mullen, and their co-workers, the NMR structures (16, 19) and the results of site-directed mutagenesis of the 8-kDa domain (14), were used to illustrate the map. According to Prasad et al. (14), the site-directed mutants of Phe-25, Lys-35, Lys-60, or Lys-68 were impaired template DNA binding activity. Since the fatty acids bind to the ssDNA-binding region of the 8-kDa domain and compete for binding with template DNA as shown in Fig. 1, two of the maps were compared. In Fig. 6B, the residues (Gln-31, Asn-37, Arg-40, Lys-41, Ala-57, Glu-58, Lys-60, Gly-66, Lys-68, Glu-71, Lys-72, Glu-75, Ala-78, Leu-82, Arg-83, and Leu-85) of the p(dT)₈ interaction interface from the HMQC NMR experiment (16) are shown. As shown in Fig. 6, A and B, the only site shifted not only by fatty acid binding, but by ssDNA (i.e. p(dT)₈) binding was Lys-35 in the -type loop including helix-1 and helix-2. The region II shown in Fig. 6A appears to have an important role in the fatty acid effect. The fatty acids probably compete with template DNA at the residue Lys-35 and bind to the site, which subsequently inhibits the ssDNA binding activity on the 8-kDa domain. In the region I shown in Fig. 6A, Leu-11, His-51, and Thr-79 are different from the other DNA binding sites (Phe-25, Lys-60, and Lys-68), suggesting that the methyl end of fatty acids disturb the binding of the template DNA at region I. Lys-35 in region II is a hydrophilic amino acid, and Leu-11 and His-51 in region I are hydrophobic amino acids. The carboxyl ends of the fatty acids may, therefore, show a preference for binding to the hydrophilic site, and the other side, the methyl end, may be absorbed to the hydrophobic site. We reported previously (2, 3) that longer chain fatty acids inhibited the binding activity more strongly and that C₁₆ or shorter fatty acids have no inhibitory effect. The distance between regions I and II may be a key to explain these characteristics of the inhibition by fatty acids. As shown in Figs. 3, 4C, and 5, the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The longer chain fatty acids (over C₂₄) are expected to more tightly bind to these residues of amino acids in the 8-kDa domain.

Modeling of the Fatty Acid Interaction Interface-- -To confirm the above assumption, we performed modeling analysis using the results of NMR experiments. The results of computer simulation of the binding mode between the N-terminal 8-kDa domain and the fatty acids are shown in Fig. 7. NA (yellow line) on the 8-kDa domain (blue-white line) in Fig. 7, A and B, was bridged from Lys-35 (red line) to Leu-11 (red line), His-51 (red line), and Thr-79 (red line) and intercalated smoothly into the pocket between helix-1 and helix-2 in the -type loop. The distance between the Lys-35 hydrophilic region and Leu-11 and His-51 hydrophobic regions fit the length of U-shaped NA. On the other hand, LA (yellow line) was trapped more deeply in the pocket, although the LA binding model is basically the same as that of NA, and was further away from regions I and II than in the NA binding model (Fig. 7, C and D). The methyl end of LA may not be able to bind firmly to region I, and subsequently, a steeper U-shaped model was postulated for LA (Fig. 7, C and D). In this simulation, the fatty acid structures were modeled, but the 8-kDa domain structure was fixed. Although the residues shown by red lines seemed to be separated from the fatty acid structures (yellow lines) (Fig. 7), the fatty acid ends are thought to bind to the respective amino acid residues, and at least the Lys-35 binding area in the 8-kDa domain peptide must be strained. The unstructured segment of the 1-13 turn is comprised of the N-terminal residues and is flexible. The 79-87 turn, i.e. the unstructured linker segment, must also be structurally flexible. Therefore, when the fatty acids bound to the domain at His-51 and Lys-35, the N-terminal turn including Leu-11 and the unstructured linker segment including Thr-79 appear to be adjacent to His-51 in the 45-55 turn. The longer chain fatty acids, since they can more likely gain access to region I, can affect the tighter binding to region I. LA or shorter fatty acids are located at some distance from the site and may hardly induce the movement. We also reported previously that the saturated forms of fatty acids, for example C₁₈-stearic acid, had no inhibitory effect on pol , although then could suppress the activity of pol  (2). The carbon chain in the saturated form fatty acid molecule is linear and does not form a U-shaped curve as seen in the unsaturated form fatty acids such as LA and NA. The linear chain may not be able to intercalate between helix-1 and helix-2 in the -type loop and thus cannot inhibit pol  activity.

View larger version (116K): [in this window] [in a new window]   Fig. 7.   Simulation of the fatty acid interaction interface on the N-terminal 8-kDa domain of rat DNA polymerase  (Protein Data Base code 1BNO). Interactions between the 8-kDa domain and NA (A and B) or LA (C and D) are shown. The x-ray crystal structure of the N-terminal 8-kDa domain of pol  is shown in blue-white. The amino acid residues Leu-11, Lys-35, His-51, and Thr-79, which were significantly shifted as the cross-peaks from HMQC NMR experiments, are depicted in red. The fatty acids are indicated in yellow. This figure was displayed using Mol Graph (Daikin Industry Ltd.).

In conclusion, the lack of an effect of shorter chain fatty acids, the positive relationship between longer carbon chain length and tighter binding, and the configuration effectiveness on pol  can be explained by our model.

The inhibitory effects of fatty acids on DNA polymerase activity occur by binding between the 8-kDa domain and the fatty acid as a 1:1 complex, and this binding can be released by nonionic detergents (2, 3). The fatty acids are present on the internal surface of the cytoplasmic membranes. These observations suggest that the inhibitory effect of fatty acids on DNA polymerase activity occurs in vivo and is reversibly controlled by binding to or release of the DNA polymerase from the fatty acids, perhaps on the membranes. These observations may help in determining the mechanisms of control of these enzymes in vivo.

	ACKNOWLEDGEMENT

We are grateful to Dr. A. Matsukage of Aichi Cancer Center Research Institute for his advice and encouragement for this work. We are also grateful to K. Suetsuna of Daikin Industry Ltd. for the technical support of computer graphics. We thank Dr. H. Taguchi of our department for helpful discussion, and A. Ogawa, Y. Fukuyo, and S. Tojo of our department for their helpful support.

	FOOTNOTES

^* This work partly was supported by the Sasakawa Scientific Research Grant (to Y. M.) from The Japan Science Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 81-471-24-1501. Fax: 81-471-23-9767; E-mail: kengo@rs.noda.sut.ac.jp

	ABBREVIATIONS

The abbreviations used are: pol, DNA polymerase (EC 2.7.7.7); NA, nervonic acid; LA, linoleic acid; ssDNA, single strand deoxyribonucleic acid; HMQC, heteronuclear multiple-quantum correlated spectroscopy; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

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