A 17S MULTIPROTEIN FORM OF MURINE CELL-DNA POLYMERASE MEDIATES POLYOMAVIRUS DNA-REPLICATION IN-VITRO

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of c entrifugation, polyethylene glycol precipitation, and ion-exchange chr omatography steps. Proteins and enzymatic activities associated with t his mouse cell multiprotein form of DNA polymerase include the DNA pol ymerases alpha and delta, DNA primase, proliferating cell nuclear anti gen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and I I. The sedimentation coefficient of the multiprotein form of DNA polym erase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is ma intained after treatment with detergents, salt, RNase, DNase, and afte r chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interacti on with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitr o polyomavirus DNA synthesis are present within the isolated 17S multi protein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Compl ex (MRC) based an the fractionation and chromatographic profiles of th e individual proteins found to co-purify with the complex. (C) 1994 Wi ley-Liss, Inc.