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- W2017285840 abstract "We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [α-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis. We demonstrate dependence on DNA pol δ and the pol α/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions. In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p. Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2." @default.
- W2017285840 created "2016-06-24" @default.
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- W2017285840 date "1998-08-01" @default.
- W2017285840 modified "2023-09-23" @default.
- W2017285840 title "Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template 1 1Edited by M. Yaniv" @default.
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- W2017285840 doi "https://doi.org/10.1006/jmbi.1998.1973" @default.
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