Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases.
Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA. By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro. Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro. It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer. Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues. Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained. Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension. The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex. Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers. That distamycin increases the thermal stability of [32P](dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated [32P](dA)9 primer. It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus.
Levy
A
A
Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
Weisman-Shomer
P
P
Fry
M
M
D000249:Adenosine Monophosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004214:Distamycins / Q000494:pharmacology*; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011071:Poly T / Q000378:metabolism*; D011067:Poly dA-dT / Q000096:biosynthesis*; D011089:Polydeoxyribonucleotides / Q000096:biosynthesis* / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology*; D013696:Temperature; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; D004214:Distamycins; D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D011758:Pyrroles; C056750:factor D (liver); C030570:oligodeoxyadenylic acid; D011071:Poly T; D011067:Poly dA-dT; D000249:Adenosine Monophosphate; D004247:DNA; D004256:DNA Polymerase I