Multiple forms of DNA polymerase from the thermo-acidophilic eubacterium Bacillus acidocaldarius: purification, biochemical characterization and possible biological role.
Two DNA polymerase isoenzymes, called DpA and DpB on the basis of their elution order from DEAE cellulose, were purified to homogeneity from the thermo-acidophilic eubacterium Bacillus acidocaldarius. The enzymes are weakly acidophilic proteins constituted by a single subunit of 117 and 103 kDa respectively. DpA and DpB differ in thermostability, in thermophilicity, in sensitivity to assay conditions and in resistance to sulphydryl-group blocking agents such as N-ethylmaleimide and p-hydroxymercuriobenzoate. They differ also in synthetic template-primer utilization, in the apparent Km for dNTPs and in processivity. In particular, DpA utilizes more effic iently synthetic templates-primers such as poly(dA).poly(dT), poly(dT). (rA)12-18 and poly(rA).(dT)12-18 and presents a greater tendency to accept dNTP analogues modified in the sugar or in the base ring, such as cytosine beta-d-arabinofuranoside 5'-triphosphate, 2',3'-dideoxyribonucleosides 5'-triphosphate, butylphenyl-dGTP and digoxigenin-conjugated dUTP. In addition, DpA presents an exonuclease activity that preferentially hydrolyses DNA in the 5'-3' direction, whereas DpB lacks this activity. The possible biological role of the enzymes is discussed.
De Falco
M
M
Istituto di Biochimica delle Proteine ed Enzimologia del Consiglio Nazionale delle Ricerche, Via Toiano 6, 80072 Arco Felice, Naples, Italy.
Grippo
P
P
Rossi
M
M
Orlando
P
P
D001407:Bacillus / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007525:Isoelectric Focusing; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D004259:DNA-Directed DNA Polymerase