The positioning and dynamics of origins of replication in the budding yeast nucleus

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridi zation and green fluorescent protein (GFP)-tagged chromosomal domains. In b oth cases, origin position was determined with respect to the nuclear envel ope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localizat ion does not necessarily persist in S phase. In contrast, early firing orig ins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal con text in G1 phase, it remains late-firing but moves rapidly away from the te lomere with which it was associated, suggesting that the positioning of yea st chromosomal domains is highly dynamic. This is confirmed by time-lapse m icroscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucl eus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both auto nomous and mobile within the nucleus.