Updated project metadata. The chromatin at origins of replication is the template for DNA replication initiation in eukaryotic genomes. However, whether the chromatin composition instructs individual origins to fire early-efficiently (EE) or late-inefficiently (LI) remains to be investigated. Here, we used site-specific recombination and a single-locus chromatin isolation approach to purify selected EE and LI replication origins from chromosome III of Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of defined ~1kb native chromatin regions surrounding the EE and LI replication start sites. We confirm expected origin interactors but also find unexpected interactions, such as the Ask1 subunit of the kinetochore-associated DASH complex. Strikingly, we show that Ask1 is a critical factor in the replication timing control of specific origins in yeast. These results highlight that our unbiased approach can specifically identify the functionally-relevant proteome at a single-copy locus of interest and provide a detailed view of histone modification and protein interaction networks on replication origin chromatin. The current dataset contains proteomic data used for the histone PTM analysis.