High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells

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Abstract
A new method, utilizing selective photodegradation of 5-bromo-deoxyuridine (BUdR)-substituted DNA and flow cytometry, has been developed for analyzing the timing of replication of specific DNA sequences. Chemically synchronized Chinese hamster ovary cells were given a pulse of the deoxythymidine analogue, BUdR, at different times during S phase, and flow sorted according to DNA content, before DNA isolation. Newly-replicated, unifilarly BUdR-substituted DNA was selectively degraded by treatment with 33258 Hoechat plus near UV light followed by SI nuclease digestion; the resistant DNA was analyzed for its content of 18s and 28s rDNA or dihydrofolate reductase (DHFR) sequences via Southern blot analysis. Both the rDNA and DHFR sequences were found to replicate almost entirely during the first quarter of S phase. The approach described should have general utility for analyzing replication kinetics of specific DNA sequences in mammalian cells.