TY - JOUR
T1 - Efficient long DNA gap-filling in a mammalian cell-free system
T2 - A potential new in vitro DNA replication assay
AU - Nakao, Seiki
AU - Zhang, Sufang
AU - Vaara, Markku
AU - Syväoja, Juhani E.
AU - Lee, Marietta Y.
AU - Tsurimoto, Toshiki
AU - Karran, Peter
AU - Oda, Shinya
N1 - Funding Information:
This study was supported by a Grant-in-aid for Cancer Research from the Ministry of Health, Labour and Welfare and grants from the Ministry of Education, Science, Sports and Culture of Japan .
PY - 2013/2
Y1 - 2013/2
N2 - 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.
AB - 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.
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U2 - 10.1016/j.biochi.2012.09.031
DO - 10.1016/j.biochi.2012.09.031
M3 - Article
C2 - 23063694
AN - SCOPUS:84872779393
SN - 0300-9084
VL - 95
SP - 320
EP - 328
JO - Biochimie
JF - Biochimie
IS - 2
ER -