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Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization


, : Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization. Journal of Biochemistry 91(2): 607-618

A mouse DNA polymerase accompanied by a novel RNA polymerase activity and its specific protein factor (stimulating factor) were purified from Ehrlich ascites tumor cells and partially characterized. The DNA polymerase was thought to be a subspecies of DNA polymerase alpha, and to be accompanied by or copurified with RNA polymerase activity capable of synthesizing RNA, which was probably utilized as a primer for subsequent DNA polymerization on a template of poly(dT) or poly(dC). This coupled reaction by RNA and DNA polymerase activities required the stimulating factor in addition to ribo- and deoxyribonucleotide substrates, although the degree of requirement depended on the kind of template and ribonucleotide substrate: the activity to incorporate dATP with poly(dT) plus ATP depended greatly on the stimulating factor, while the activity to incorporate dGTP with poly(dC) did not when GTP was added at high concentrations. GDP could be substituted for GTP, but the activity with poly(dC) plus GDP depended largely on the stimulating factor. Involvement of known RNA polymerases in the activity with poly(dT) was excluded, because addition of purified mouse RNA polymerases I and II had no effect on the incorporation of dATP, and alpha-amanitin (100 micrograms/ml) did not inhibit the incorporations of dATP and ATP. Analysis of the inhibition by the nucleotide analog 2',3'-dideoxynucleoside 5'-triphosphate (ddNTP) further supported the involvement of new RNA polymerase; ddNTPs inhibited the activities with poly(dT) and poly(dC) significantly more than RNA polymerases I and II or DNA polymerase alpha activity with poly(dT) . oligo(rA) and poly(dC) . oligo(dG) as template. Lineweaver-Burk analysis of the inhibitions showed that ddATP inhibited competitively with respect to ATP, and ddGTP inhibited competitively with respect to GDP but noncompetitively with respect to GTP.

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PMID: 7068578


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