The eukaryotic genome is highly packed with histones to form chromatin. The basic building unit of chromatin is the nucleosome, which consists of a histone octamer core, wrapped by 147 base pairs of DNA. The nature of the nucleosome structure presents a formidable barrier for DNA-related processes, especially for DNA replication. Therefore, the chromatin will undergo dramatic dynamics during replication, involving disassembly of old nucleosomes and distribution of both new and old histones to form nucleosomes onto both daughter DNA strands. These nucleosome dynamics suggest a challenge for the maintenance of histone density and epigenetic inheritance in the wake of DNA replication. Chromatin Assembly Factor-1 (CAF-1) is a conserved histone chaperone that directly interacts with the replication machinery via the polymerase processivity factor PCNA, and is involved in assembling nucleosomes behind the DNA replication fork. CAF-1 is essential for multicellular eukaryotes, while deletion of CAF-1 in yeast is not lethal, but results in increased sensitivity to DNA damage and aberrant telomeric silencing. Despite the significance of this histone chaperone, the structural organization of this complex remains largely unknown, and thus the mechanism underlying CAF-1-mediated nucleosome assembly is elusive. In this study, we identified the key peptides involved in CAF-1 subunit assembly by performing HDX-MS analysis followed by site-directed mutagenesis studies, which were confirmed by yeast genetic studies. This structural information allows us to further characterize functional domains within CAF-1, and provides unprecedented details for future structural studies using crystallization and/or cryo-EM. This work also shows how histones H3-H4 are bound by CAF-1, and how this histone binding regulates the nucleosome assembly activity by CAF-1. We also show that DNA is acting as a bridge to bring two histone-bound CAF-1 together, thereby promoting (H3-H4)2 tetramer formation as well as the tetramer hand-off between CAF-1 and DNA, resulting in the formation of tetrasome ((H3-H4)2 tetramer wrapped with DNA), the initial step for nucleosome assembly. Overall, this study provides a mechanistic explanation for efficient nucleosome assembly by CAF-1 following DNA replication, and highlights a direct nucleosome assembly mechanism by a histone chaperone for the first time. Moreover, the concerted mechanism of CAF-1-mediated nucleosome assembly suggests that two H3-H4 dimers are brought together right before the (H3-H4)2 tetramer deposition onto DNA, shedding light on the future directions of epigenetic maintenance regulation during replication.