Abstract
DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.
Highlights
DNA synthesis in eukaryotes must be performed with absolute precision as any defects compromise genetic integrity
Sphase progression is defined by the spatial organization of DNA foci In HeLa cells in early S phase, the template for DNA synthesis is folded into DNA foci that can be labeled with a variety of modified thymidine analogues and visualized in both living and fixed cells (Figure S1)
Different pulse and pulse-chase-pulse strategies can be used to evaluate the relationship of foci that are engaged in DNA synthesis during different intervals of S phase (Figure S2)
Summary
DNA synthesis in eukaryotes must be performed with absolute precision as any defects compromise genetic integrity. Different regions of the genome are replicated at specific times [4,5,6], perhaps as a part of a fundamental mechanism that ensures the preservation of epigenetic information [7] Within this timing program, chromatin within gene-rich chromosomal R-bands is known to begin early in S phase, before synthesis of heterochromatic G-bands takes place. Chromatin within gene-rich chromosomal R-bands is known to begin early in S phase, before synthesis of heterochromatic G-bands takes place This general structure can be revealed at low resolution, using cytological chromosome banding [8], and at higher resolution using genome-wide strategies [9,10,11,12,13,14,15]. More detailed analysis is beginning to explore how local chromatin features such as the distribution of CpG islands [14] and local chromatin accessibility [15] contribute to patterns of origin selection
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