Expression of active rat DNA polymerase .beta. in Escherichia coli

Abstract

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.

A novel DNA topoisomerase inhibitor: dehydroebriconic acid, one of the lanostane‐type triterpene acids from Poria cocos

“…We also analyzed the kinetics of compound 5, and obtained similar results (data not shown). In the kinetic analysis, poly(dA)/oligo(dT) [12][13][14][15][16][17][18] and dTTP were used as the DNA template-primer and nucleotide substrate, respectively. Double-reciprocal plots of the results show that inhibition of pol α activity by compound 6 was noncompetitive with the DNA template and the nucleotide substrate.…”

“…Similar results were observed with compound 5: the pol α inhibition was non-competitive with both DNA template-primer and dTTP, but the pol β inhibition was competitive with both DNA template-primer and dTTP. When activated DNA was used as the template-primer DNA instead of synthesized DNA (i.e., poly(dA)/oligo(dT) [12][13][14][15][16][17][18] ), the inhibitory modes of the compounds were unchanged (data not shown). The triterpene acids may interact with or affect both of the binding sites on pol β, thereby decreasing its affinity for the DNA template and substrate, whereas they may bind or interact with a domain distinct from the template or substrate binding sites on pol α.…”

“…Activities of DNA polymerases were measured by the methods described previously. 24,25) The substrates of DNA polymerases used were poly(dA)/oligo(dT) [12][13][14][15][16][17][18] and dTTP as template-primer DNA and nucleotide substrate, respectively. One unit of each DNA polymerase activity was defined as the amount of enzyme that catalyzes the incorporation of 1 nmol of deoxyribonucleotide triphosphate (i.e., dTTP) into the synthetic template-primers (i.e., poly(dA)/oligo(dT) [12][13][14][15][16][17][18] , A/T=2/1) in 60 min at 37°C under the normal reaction conditions for each of the enzymes.…”

“…Therefore, information concerning the structural characteristics of these inhibitors may provide valuable insight for the design of new anti-cancer agents. [12][13][14][15][16][17][18] were purchased from Amersham Biosciences (Buckinghamshire, UK). Supercoiled pUC19 plasmid DNA was obtained from TaKaRa (Tokyo).…”

.BETA.-Sitosteryl (6'-O-linoleoyl)-glucoside of Soybean (Glycine max L.) Crude Extract Inhibits Y-Family DNA Polymerases

“…Recombinant rat pol β was purifi ed from Escherichia coli JMpβ5 as described by Date et al 11 . The human pol γ catalytic gene was cloned into pFastBac Invitrogen Japan K.K., Tokyo Japan .…”