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Human cell DNA replication is mediated by a discrete multiprotein complex
Author(s) -
Jiang Hai Yan,
Hickey Robert J.,
AbdelAziz Waleed,
Tom Timothy D.,
Wills Philip W.,
Liu Jianying,
Malkas Linda H.
Publication year - 2002
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.10182
Subject(s) - dna clamp , dna polymerase ii , dna polymerase , eukaryotic dna replication , dna replication , microbiology and biotechnology , biology , dna polymerase delta , dna , polymerase , multiprotein complex , biochemistry , polymerase chain reaction , reverse transcriptase , gene
A discrete high molecular weight multiprotein complex containing DNA polymerase α has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion‐exchange chromatography indicates that the peak of DNA polymerase α activity co‐purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase δ, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS–PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell. J. Cell. Biochem. 85: 762–774, 2002. © 2002 Wiley‐Liss, Inc.