Eukaryotic chromosomal DNA is faithfully replicated in a complex series of 
cell-cycle-regulated events that are incompletely understood. Here we report the 
reconstitution of DNA replication free in solution with purified proteins from 
the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated 
bidirectional origin activation; synthesis of leading and lagging strands by the 
three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical 
maturation of Okazaki fragments into continuous daughter strands. We uncover a 
dual regulatory role for chromatin during DNA replication: promoting origin 
dependence and determining Okazaki fragment length by restricting Pol δ 
progression. This system thus provides a functional platform for the detailed 
mechanistic analysis of eukaryotic chromosome replication.