DNA binding domain of Escherichia coli DNA polymerase I: identification of arginine-841 as an essential residue

To identify the DNA binding site(s) in Escherichia coli DNA polymerase I (pol I) (Klenow fragment), we have used an active-site-directed reagent, phenylglyoxal (PG), which specifically reacts with arginine residues. Preincubation of DNA pol I with PG resulted in the loss of polymerase, 3'-5'-exonuclease, and DNA binding functions. Furthermore, the presence of DNA but not deoxynucleoside triphosphates protected the enzyme from inactivation. Labeling studies with [7-14C]PG indicated that two arginine residues were modified per mole of enzyme. In order to locate the site of PG modification, we digested the PG-treated enzyme with trypsin and V-8 protease. The resulting peptides from each digest were then resolved on reverse-phase hydrophobic columns. An appearance of a new peptide peak was observed in both tryptic and V-8 protease digests. Since inclusion of template-primer during PG modification of enzyme blocks the appearance of these peaks, these peptides were concluded to represent the template-primer binding domain of pol I. Indeed, the extent of inactivation of enzyme by PG treatment correlated very well with the quantitative increase in the new tryptic peptide peak. Amino acid composition analysis of both tryptic peptide and V-8 peptide revealed that the two peptides were derived from the same general region; tryptic peptide spanned between residues 837 and 857 while V-8 peptide spanned between residues 841 and 870 in the primary sequence of pol I. Sequence analysis of tryptic peptide further identified arginine-841 as the site of PG modification, which implicates this residue in the DNA binding function of pol I.     

Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I

We have labeled the large fragment of Escherichia coli DNA polymerase I (Pol I) with pyridoxal 5'-phosphate, a substrate binding site directed reagent for DNA polymerases [Modak, M. J. (1976) Biochemistry 15, 3620-3626]. A covalent attachment of pyridoxal phosphate to Pol I results in the loss of substrate binding as well as the polymerase activity. The inactivation was found to be strictly dependent on the presence of a divalent metal ion. Four moles of pyridoxal phosphate was found to react per mole of the enzyme, while in the presence of substrate deoxynucleoside triphosphate only 3 mol of pyridoxal phosphate was bound. To identify the substrate-protected site on the enzyme, tryptic peptides from enzyme labeled with pyridoxal phosphate and tritiated borohydride, in the presence and absence of substrate, were resolved on a C-18 reverse-phase column. A single peptide containing the substrate-protected site was identified and further purified. The amino acid composition and sequence analysis of this peptide revealed it to span residues 756-775 in the primary acid sequence of Pol I. Lys-758 of this sequence was found to be the site of the pyridoxal phosphate reaction. It is therefore concluded that Lys-758 is the site of binding for the metal chelate form of nucleotide substrates in E. coli DNA polymerase I.     

Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli. A direct photoaffinity labeling study

The physical basis of ATP binding and activation of DNA polymerase III holoenzyme was studied by an ultraviolet irradiation cross-linking technique. ATP and dATP were photocrosslinked to the alpha, tau, gamma, and delta subunits of holoenzyme; photocrosslinking of dATP was competitively inhibited by ATP. No photocrosslinking was observed with GTP or CTP, nor did GTP, CTP, or UTP inhibit cross-linking of ATP. ADP and adenosine 5'-O-(3-thio)-triphosphate, both potent inhibitors of ATP activation of holoenzyme, inhibited cross-linking of ATP to tau, gamma, and delta subunits, but not to the alpha subunit, suggesting that one or more of these subunits are ATP (or dATP)-binding sites. Photocrosslinking of dTTP to the ATP-activated holoenzyme was exclusively to the epsilon subunit, the dnaQ ( mutD ) gene product; dCTP and dGTP were not photocrosslinked to any subunit. Binding of dTTP was enhanced by ATP, but by no other nucleotide (or deoxynucleotide). This binding of dTTP to epsilon, a subunit likely responsible for regulation of proofreading by the holoenzyme, may function in the control of the fidelity of replication.     

Affinity labeling of Escherichia coli DNA polymerase I by 5'-fluorosulfonylbenzoyladenosine. Identification of the domain essential for polymerization and Arg-682 as the site of reactivity

Preincubation of Escherichia coli DNA polymerase I (pol I) with 5'-fluorosulfonylbenzoyladenosine (5'-FSBA) results in an irreversible inactivation of DNA polymerase activity with concomitant covalent binding of 5'-FSBA to enzyme. pol I-associated 3'-5' exonuclease activity, however, remains unaffected. Kinetic studies of inactivation indicate that the degree of inactivation is directly proportional to the concentration of 5'-FSBA and increases linearly with time. The presence of the metal chelate form of dNTP substrates or template primer, but not the template or primer alone, protects the enzyme from inactivation by 5'-FSBA. A complete inactivation of polymerase activity occurs when 2 mol of 5'-FSBA are covalently linked to 1 mol of enzyme, suggesting two sites of modification. Tryptic peptide mapping of 5'-FSBA-treated enzyme revealed the presence of two distinct peptides containing the affinity label, confirming the presence of two reactive sites in the enzyme. However, we find that only one of the two sites is essential for the polymerase activity since, in the presence of substrate dNTP or template primer during preincubation of enzyme with 5'-FSBA, incorporation of the affinity label is reduced by only 1 mol. Peptide analysis of dNTP or template primer-protected enzyme further revealed that a peptide eluting at 35 min from the C-18 matrix was protected from the 5'-FSBA reaction. It is therefore concluded that this peptide contains the domain essential for polymerase activity. Staphylococcus aureus V-8 protease digestion, amino acid composition, and sequence analysis of this peptide revealed this domain to span residues 669 to 687 in the primary amino acid sequence of pol I, and arginine 682 was found to be the site of 5'-FSBA reactivity.     

Photoaffinity labeling of the nucleotide binding site of actin

Rabbit skeletal muscle actin was photoaffinity-labeled by the nucleotide analogue 8-azidoadenosine 5'-triphosphate. In both G-actin and F-actin about 25% covalent incorporation was achieved. The labeled actins were digested with cyanogen bromide, and the labeled peptides were isolated and sequenced. In F-actin the label was bound primarily to Lys-336, while in G-actin the label was bound to Lys-336 or to Trp-356. The results indicate that the nucleotide binding site is near the phalloidin binding site of actin [Vanderkerckhove, J., Deboben, A., Nassal, M., & Wieland, T. (1985) EMBO J. 4, 2815-2818]. The binding of the azido group to Trp-356 in G-actin but not in F-actin may indicate that a change in the conformation of actin occurs in this region.     

Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate

A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.     

Highly selective affinity labeling of the primer-binding site of E. coli DNA polymerase I

Membrane-associated phosphoinositidase C activity has been identified in Dictyostelium discoideum using phosphatidylinositol 4,5-bisphosphate as exogenous substrate. Maximal activity was observed with 0.4 mM phosphatidylinositol 4,5-bisphosphate at pH 7.0. The enzyme was stimulated

by micromolar concentrations of free calcium with maximal activity at 100 μM.     

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [125I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.     

Template primer-dependent binding of 5'-fluorosulfonyl-benzoyldeoxyadenosine by Escherichia coli DNA polymerase I. Identification of arginine 682 as the binding site and its implication in catalysis

We have labeled a template primer-dependent substrate deoxynucleoside triphosphate binding domain in Escherichia coli DNA polymerase I using an affinity labeling analogue of dATP, the 5'-fluorosulfonylbenzoyldeoxyadenosine (FSBdA). Using enzyme-template primer complex as a test system, we find that FSBdA-mediated inactivation occurs only when the template in the enzyme-template primer complex is poly(dT).(dA)10. A ribonucleotide analogue, 5'-fluorosulfonylbenzoyladenosine (FSBA) is not an effective inactivator under these conditions. In the absence of template primer, however, deoxyribonanalogue (FSBdA) irreversibly inactivates polymerase activity with characteristics similar to those reported for FSBA (Pandey, V.N., and Modak, M.J. (1988) J. Biol. Chem. 263, 6068). Binding stoichiometric studies in the presence and absence of template primer revealed that only 1 mol of FSBdA is incorporated per mol of enzyme which results in complete inactivation. The site of FSBdA action was investigated by comparative tryptic peptide mapping, followed by amino acid composition analysis of the modified peptide. Arginine 682 was found to be the target of FSBdA reactivity. We therefore conclude that the domain containing Arg-682 plays a major role in template-dependent dNTP binding and polymerization.     

Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment). Evidence that binding protein inhibits trans-lesion synthesis by polymerase I

The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E. coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis. The two polymerase enzymes displayed differential responses to the addition of SSB. T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used. On the other hand, E. coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates. This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis. Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.     

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.     

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8-azido-AMP

The photoreactive AMP analog, 8-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the Km for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 microM and 1.5 microM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Photolabeling of AMP-free dehydratase with 8-azido-[2-3H]AMP resulted in a time and concentration-dependent enzyme inactivation and concomitant incorporation of 8-azido-AMP into protein. At low 8-azido-AMP concentrations, incorporation of about 1 mol 8-azido-AMP/mol dehydratase tetramer was correlated with almost complete inactivation of the enzyme. The presence of AMP in the photolabeling reaction greatly reduced the extent of enzyme inactivation and 8-azido-AMP binding. Ultraviolet irradiation with 20 microM 3H-labeled 8-azido-AMP revealed one tryptic peptide, Thr230-Thr-Gly-Thr-Leu-Ala-Asp-Gly-Cys-Asp-Val-Ser-Arg242, with bound radioactivity. This peptide, labeled at low concentration of 8-azido-AMP, most likely represents the AMP-binding region on the dehydratase molecule.     

Interaction of Escherichia coli DNA polymerase I with azidoDNA and fluorescent DNA probes: identification of protein-DNA contacts

The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.     

Spacing mutations between the Escherichia coli pBAD RNA polymerase binding site and the araC (I) induction site

Mutations in the Escherichia coli promoter PBAD have been constructed which alter the spacing of the adjacent RNA polymerase and araC inducer protein binding sites. While deletion of a single base-pair or small insertions do not detectably affect araC protein binding to DNA and they do not alter the conserved sequence of the RNA polymerase binding site, stimulation of PBAD in vivo is greatly reduced. The experiments suggest that the distance or angle between the two proteins on the DNA is critical for promoter function.     

Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase

A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.     

The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable

Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val(700)-Arg(712) by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp(705) can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild-type motif A sequence. As an example, we established that the naturally occurring Ile(709) has a previously undocumented role in supporting sugar discrimination.     

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe

Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)