Efficient long DNA gap-filling in a mammalian cell-free system: a potential new in vitro DNA replication assay.
In vitro assay of mammalian DNA replication has been variously approached. Using gapped circular duplex substrates containing a 500-base single-stranded DNA region, we have constructed a mammalian cell-free system in which physiological DNA replication may be reproduced. Reaction of the gapped plasmid substrate with crude extracts of human HeLaS3 cells induces efficient DNA synthesis in vitro. The induced synthesis was strongly inhibited by aphidicolin and completely depended on dNTP added to the system. In cell extracts in which PCNA was depleted step-wise by immunoprecipitation, DNA synthesis was accordingly reduced. These data suggest that replicative DNA polymerases, particularly pol delta, may chiefly function in this system. Furthermore, DNA synthesis is made quantifiable in this system, which enables us to evaluate the efficiency of DNA replication induced. Our system sensitively and quantitatively detected the reduction of the DNA replication efficiency in the DNA substrates damaged by oxidation or UV cross-linking and in the presence of a potent chain terminator, ara-CTP. The quantitative assessment of mammalian DNA replication may provide various advantages not only in basic research but also in drug development.
Cancer Genetics Laboratory, Clinical Research Institute, National Kyushu Cancer Center, Fukuoka 811-1395, Japan.
Seiki
S
Nakao
Sufang
S
Zhang
Markku
M
Vaara
Juhani E
JE
Syväoja
Marietta Y
MY
Lee
Toshiki
T
Tsurimoto
Peter
P
Karran
Shinya
S
Oda
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001085:Arabinofuranosylcytosine Triphosphate / Q000494:pharmacology; D001681:Biological Assay; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D010957:Plasmids / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D001085:Arabinofuranosylcytosine Triphosphate; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase