Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil

Abstract

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.