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Further characterization of the human cell multiprotein DNA replication complex
Author(s) -
Applegren Nancy,
Hickey Robert J.,
Kleinschmidt Ann M.,
Zhou Qiqi,
Coll Jennifer,
Wills Philip,
Swaby Ramona,
Wei Yuetong,
Quan Jiang Yuan,
Lee Marietta Y. W. T.,
Malkas Linda H.
Publication year - 1995
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240590111
Subject(s) - dna polymerase ii , eukaryotic dna replication , dna polymerase , dna clamp , biology , dna replication , dna polymerase delta , microbiology and biotechnology , replication factor c , dna polymerase i , primase , control of chromosome duplication , replication protein a , proliferating cell nuclear antigen , dna , biochemistry , dna binding protein , reverse transcriptase , polymerase chain reaction , gene , transcription factor
Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication‐competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co‐purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase α, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA‐dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q‐Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin‐specific and large T‐antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362–6374.] In this report we describe the further characterization of the human cell replication‐competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co‐purify with the MRC have been identified. These proteins include: DNA polymerase δ, RF‐C, topoisomerase II, DNA ligase I, DNA helicase, and RP‐A. The replication requirements, replication initiation 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 model 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 include 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 provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells. © 1995 Wiley‐Liss, Inc.