FURTHER CHARACTERIZATION OF THE HUMAN CELL MULTIPROTEIN DNA-REPLICATION COMPLEX

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a repl ication-competent multiprotein form of DNA polymerase isolated from hu man (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase inc luded: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PC NA, and a DNA-dependent ATPase. The multiprotein form of the human cel l DNA polymerase was further purified by Q-Sepharose chromatography fo llowed by glycerol gradient sedimentation and was shown to be fully co mpetent to support origin-specific and large T-antigen dependent simia n virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Bio chemistry 29:6362-6374]. In this report we describe the further charac terization of the human cell replication-competent multiprotein form o f DNA polymerase designated MRC. Several additional DNA replication pr oteins that co-purify with the MRC have been identified. These protein s include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication ini tiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a mode l to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to incl ude the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results p rovide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells. (C) 1995 Wiley-Liss, Inc.