NYMC Faculty Publications

Efficient Long DNA Gap-Filling in a Mammalian Cell-Free System: A Potential New in Vitro DNA Replication Assay

Author Type(s)

Faculty

DOI

10.1016/j.biochi.2012.09.031

Journal Title

Biochimie

First Page

320

Last Page

328

Document Type

Article

Publication Date

2-1-2013

Department

Biochemistry and Molecular Biology

Keywords

Animals, Aphidicolin, Arabinofuranosylcytosine Triphosphate, Biological Assay, Cell-Free System, DNA Replication, DNA, Single-Stranded, DNA-Directed DNA Polymerase, HeLa Cells, Humans, Kinetics, Nucleic Acid Synthesis Inhibitors, Plasmids, Proliferating Cell Nuclear Antigen

Disciplines

Medicine and Health Sciences

Abstract

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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