Replication of polyoma DNA in isolated nuclei: III. The nucleotide sequence at the RNA—DNA junction of nascent strands

Abstract

Short fragments consisting of about 100 to 140 deoxyribonucleotides serve as intermediates in the elongation of polyoma DNA. In nuclei isolated from polyoma-infected 3T6 mouse fibroblasts these fragments are initiated by stretches of RNA. We investigated the nature of the ribo- and deoxyribonucleotides at the RNA-DNA link. DNA was synthesized in vitro from each of the four α- 32P-labelled deoxynucleoside triphosphates, the nascent strands were hydrolysed with alkali and the transfer of isotope to ribonucleotides was studied after fractionation of strands according to size. Each strand contained on the average one RNA-DNA link at the 5′ end of DNA. All four common ribo- and deoxyribonucleotides were present at the RNA-DNA link with close to equal frequency, irrespective of chain length or incubation time. In a second approach, daughter strands synthesized in vivo were treated with alkali and the 5′-OH ends of DNA liberated were 32P-labelled using polynucleotide kinase. All four deoxynucleotides were labelled by this treatment confirming the corresponding results of the in vitro experiments. During the discontinuous synthesis of polyoma DNA the switch from RNA to DNA synthesis is thus not effected by a specific sequence at the RNA-DNA junction, in contrast to Escherichia coli where the sequence p(rPy)p(dC)p was reported.