PHOSPHORYLATION OF OKAZAKI-LIKE DNA FRAGMENTS IN MAMMALIAN-CELLS AND ROLE OF POLYAMINES IN THE PROCESSING OF THIS DNA

In mammalian cells DNA synthesis is more complicated than in prokaryot es and less well understood. Here we incubated intact mammalian cells (polyamine auxotrophic Chinese hamster ovary cells and primary human f ibroblasts) with [P-32]orthophosphate and found that, besides high mol ecular weight DNA, a species of low molecular weight DNA, similar to 4 50 bp in size, became efficiently labeled, The short DNA was labeled f irst, and in pulse-chase experiments the labeling was transient. The i solated small DNA fragments (RNase A-treated) were phosphorylated by T 4 polynucleotide kinase specific for polynucleotides with 5'-OH ends. A polynucleotide kinase phosphorylating these DNA pieces was also dete cted in nuclear extracts of the cells. Treatment with alkaline phospha tase removed most of the P-32 label incorporated into the small DNA in vivo. Labeling with deoxyribonucleosides did not reveal these fragmen ts. We hypothesize that the low molecular weight DNA represents Okazak i fragments and that the mammalian DNA replication machinery includes a polynucleotide kinase phosphorylating the 5'-termini of Okazaki frag ments. This would imply a novel step in DNA synthesis. We also show th at depriving cells of polyamines reversibly blocks synthesis of high m olecular weight DNA and leads to accumulation of the short DNA pieces, suggesting a role for polyamines in joining the Okazaki fragments.