Association of 3′ → 5′ exodeoxyribonuclease activity with DNA replitase complex from S-phase Chinese hamster embryo fibroblast cells

Abstract

DNA replitase has been described as a complex of enzymes/proteins that are associated with both DNA precursor biosynthesis and DNA replication in mammalian cells [ Reddy, G. P. V., and Pardee, A. B. (1980) Proc. Natl. Acad. Sci. USA 77, 3312–3316]. We demonstrate for the first time a 3′ → 5′ exodeoxyribonuclease activity is associated with the replitase complex. As much as 60% of this exonuclease activity was similar to that associated with DNA polymerase δ based upon its sensitivity to inhibition by GMP and by butyl-phenyl-deoxyguanosine triphosphate (BuPdGTP). Association of 3′ → 5′ exonuclease activity with the DNA polymerase in the replitase complex was also demonstrated by analyzing dTTP turnover to dTMP in an in vitro DNA polymerase assay system. The DNA polymerase activity in replitase complex exhibited a sensitivity to BuPdGTP which both was similar to that of DNA replication in permeable cells and was intermediate between the BuPdGTP inhibition of purified DNA polymerases α and δ. These studies suggest that the replitase complex contains 3′ → 5′ exonuclease activity associated with the DNA polymerase activity responsible for nuclear DNA replication in mammalian cells. Further studies are required to determine if these activities are at least partially attributed to DNA polymerase δ.