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A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro
Author(s) -
Wu Yan,
Hickey Robert,
Lawlor Kenneth,
Wills Philip,
Yu Fang,
Ozer Harvey,
Starr Robyn,
Quan Jiang Yuan,
Lee Marietta,
Malkas Linda
Publication year - 1994
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.240540105
Subject(s) - dna polymerase , dna clamp , dna polymerase ii , biology , eukaryotic dna replication , microbiology and biotechnology , dna replication , dna polymerase i , multiprotein complex , proliferating cell nuclear antigen , dna polymerase delta , primase , dna , biochemistry , reverse transcriptase , polymerase chain reaction , gene
We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion‐exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases α and δ, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52‐cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction 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 vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian M ultiprotein DNA R eplication C omplex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co‐purify with the complex.