Genome stability requires one, and only one, DNA duplication at each S phase. The mechanisms preventing origin firing on newly replicated DNA are well documented, but much less is known about the mechanisms controlling the spacing of initiation events(2,3), namely the completion of DNA replication. Here we show that origin use in Chinese hamster cells depends on both the movement of the replication forks and the organization of chromatin loops. We found that slowing the replication speed triggers the recruitment of latent origins within minutes, allowing the completion of S phase in a timely fashion. When slowly replicating cells are shifted to conditions of fast fork progression, although the decrease in the overall number of active origins occurs within 2 h, the cells still have to go through a complete cell cycle before the efficiency specific to each origin is restored. We observed a strict correlation between replication speed during a given S phase and the size of chromatin loops in the next G1 phase. Furthermore, we found that origins located at or near sites of anchorage of chromatin loops in G1 are activated preferentially in the following S phase. These data suggest a mechanism of origin programming in which replication speed determines the spacing of anchorage regions of chromatin loops, that, in turn, controls the choice of initiation sites.
Adenovirus DNA integrated into the genomes of adenovirus-transformed hamster cells or of adenovirus type 12 (Ad12)-induced hamster tumor cells can be located at many different chromosomal sites. This raises the question as to whether distinct isochores of the hamster cell genome might be more accessible to recombination with adenovirus DNA. In Ad12- or Ad2-transformed hamster cell lines, and in Ad12 revertants, the investigated integrated viral DNA sequences were assigned to isochore families by analyzing DNA fractions from preparative CsCl density gradients for their buoyant densities (and, therefore, GC levels) and for the presence of viral DNA. Adenovirus DNA sequences were found in different isochores, which did not generally match the base composition of viral sequences. This is in apparent contrast to what was previously observed with retroviral integration. However, in cell lines carried in culture for many years, the viral DNA sequences might have been transposed to different isochore positions, since the host sequences flanking the viral DNA appear to have been conserved.
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