In vitro reconstitution of the end replication problem

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Altho ugh the idea has been widely accepted for explaining telomere attrition dur ing cell proliferation, it has never been directly demonstrated. In order t o take a biochemical approach to understand how linear DNA ends are replica ted, we have established a novel in vitro linear simian virus 40 DNA replic ation system. In this system, terminally biotin-labeled linear DNAs are con jugated to avidin-coated beads and subjected to replication reactions. Line ar DNA was efficiently replicated under optimized conditions, and replicati on products that had replicated using the original DNA templates were speci fically analyzed by purifying bead-bound replication products. By exploitin g this system, we showed that while the leading strand is completely synthe sized to the end, lagging strand synthesis is gradually halted in the termi nal similar to 500-bp region, leaving 3' overhangs. This result is consiste nt with observations in telomerase-negative mammalian cells and formally de monstrates the end replication problem. This study provides a basis for stu dying the details of telomere replication.