Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.
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PMID:The enzymatic basis of cyclophosphamide specificity. 302 54

Different polA mutants of Escherichia coli K-12 were exposed to ozone, X-rays and UV radiation, in order to compare the role of the various enzymatic activities of DNA polymerase I in the repair of damage caused by these agents. As was the case with radiations, the polymerase-deficient mutants were the most sensitive to ozone, followed by the 5'-3' exonuclease-deficient mutants. The 3'-5' exonuclease activity of pol I appeared to play a minor role in the repair of damage induced by all these agents.
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PMID:Sensitivity of polA mutants of Escherichia coli K-12 to ozone and radiations. 304 88

The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in Km values and a decrease in Vmax values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [3H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II).
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PMID:Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I. 306 9

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.
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PMID:Cocrystal structure of an editing complex of Klenow fragment with DNA. 319

Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel. Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II. In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity. Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity. Gel filtration indicated that the complex has a molecular mass greater than 400 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products. The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase. Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity. The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication. A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme. It degrades both single-stranded and double-stranded DNA in the 5'----3' direction.
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PMID:DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization. 327 61

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.
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PMID: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. 328 17

Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.
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PMID:Ferrate oxidation of Escherichia coli DNA polymerase-I. Identification of a methionine residue that is essential for DNA binding. 329 59

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.
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PMID:A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity. 332 25

The sequence specificity in the photoreaction of various psoralen derivatives with DNA is investigated by using DNA sequencing methodology. The 3'-5' exonuclease activity associated with T4 DNA polymerase serves as a probe to map the psoralens' photoaddition (monoadducts plus biadducts) on DNA fragments of defined sequence. This approach has already allowed us to demonstrate a strong sequence context effect on the 8-methoxypsoralen photobinding to DNA [Sage, E., & Moustacchi, E. (1987) Biochemistry 26, 3307-3314]. The psoralens studied include bifunctional derivatives [8-methoxypsoralen, 5-methoxypsoralen, and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen] and monofunctional derivatives (angelicin, 3-carbethoxypsoralen, and three pyridopsoralens). Maps of photochemical binding on two DNA fragments of the lacI gene of Escherichia coli are established for all the derivatives. These maps demonstrate the following general qualitative rules in the photoreaction of the furocoumarins with DNA: thymine residues in a GC environment are cold, adjacent thymines are better targets, 5'-TpA sites are strongly preferred versus 5'-ApT, and alternating (AT)n sequences are hot spots for photoaddition. Depending on the chemical structure of the derivatives and on their affinity for DNA, some minor differences in the binding spectrum are detected. A most interesting example is 3-carbethoxypsoralen, which specifically reacts with (AT)n sites. Our observations lead us to define two types of target sites: the "strong sites", which are preferential targets for all psoralen derivatives, and the "weak sites", which are targets only for derivatives having a high affinity for DNA. The frequency of DNA lesions is much higher in the former sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sequence specificity in photoreaction of various psoralen derivatives with DNA: role in biological activity. 340 63

We describe a new rapid method for random introduction of single-nucleotide (nt) substitutions into a small segment of cloned DNA. A DNA fragment containing a sequence to be mutagenized is inserted into a multiple cloning site sequence of a vector plasmid. The plasmid is linearized with two adjacent cuts (generating 5' and 3' protruding ends) and then synchronously and unidirectionally digested with exonuclease III (Exo III) so that the 3' termini generated are localized within the target region. A non-complementary alpha-thiophosphate nucleotide is misincorporated into the 3' terminus generated by Exo III. Since the nucleotide analogue is resistant to the 3'-5' exonuclease activity of DNA polymerase I, its misincorporation into the 3' termini is irreversible. Then, the single-stranded region is filled-in with four canonical nucleotides, and the plasmid is recircularized. This procedure was used to mutagenize a specific region of the rnpB gene of E. coli. By sequencing 72 randomly selected clones, we found that 27 clones (37.5%) had nucleotide substitutions distributed within the desired region of a 55-nt-long segment of the gene. The procedure is simple and is applicable to any DNA molecule.
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PMID:A rapid and efficient method for targeted random mutagenesis. 340 36


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