Spatial and temporal dynamics of DNA replication sites in mammalian cells

Fluorescence microscopic analysis of newly replicated DNA has revealed disc rete granular sites of replication (RS). The average size and number of rep lication sites from early to mid S-phase suggest that each RS contains nume rous replicons clustered together. We are using fluorescence laser scanning confocal microscopy in conjunction with multidimensional image analysis to gain more precise information about RS and their spatial-temporal dynamics . Using a newly improved imaging segmentation program, we report an average of similar to 1,100 RS after a 5-min pulse labeling of 3T3 mouse fibroblas t cells in early 5-phase. Pulse-chase-pulse double labeling experiments rev eal that RS take similar to 45 min to complete replication. Appropriate cal culations suggest that each RS contains an average of 1 mbp of DNA or simil ar to 6 average-sized replicons. Double pulse-double chase experiments demo nstrate that the DNA sequences replicated at individual RS are precisely ma intained temporally and spatially as the cell progresses through the cell c ycle and into subsequent generations. By labeling replicated DNA at the G(1 )/S borders for two consecutive cell generations, we show that the DNA synt hesized at early S-phase is replicated at the same time and sites in the ne xt round of replication.