Human cell DNA replication is mediated by a discrete multiprotein complex.
A discrete high molecular weight multiprotein complex containing DNA polymerase alpha 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 alpha 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 delta, 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.
Jiang
Hai Yan
HY
Department of Medicine, Hematology/Oncology Division, Indiana Cancer Research Institute, Indiana University Purdue University, Indianapolis, Indiana 46202, USA.
Hickey
Robert J
RJ
Abdel-Aziz
Waleed
W
Tom
Timothy D
TD
Wills
Philip W
PW
Liu
Jianying
J
Malkas
Linda H
LH
D002460:Cell Line; D002853:Chromatography, Liquid; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D013539:Simian virus 40 / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D004256:DNA Polymerase I; D004258:DNA Polymerase III