Abstract Our understanding of DNA replication and its relationship with chromatin structure, nuclear organization and gene transcription has advanced significantly through the development of genome-wide assays. Most genome-wide tools to study DNA replication utilize large populations of cells and report on the average behavior of the population; as such, many infrequent or stochastic events that occur as the replication fork progresses through chromatin are missed. Recently, we have developed novel methods to map DNA replication with single-molecule and single-nucleotide precision. In this proposal we will apply this new technology to understand how DNA synthesis can be completed when the replication machinery encounters obstacles. We will specifically test how RNA/DNA hybrids and G-quadruplex DNA influence the progression of DNA replication and whether DNA synthesis can be efficiently restarted downstream of an impediment. In addition to understanding the progression of replication through chromatin, we will also investigate how replication is competed during the process of termination. Using high-resolution assays, we have uncovered a critical role for the DNA helicase Rrm3 in replication termination. We will further define the role played by Rrm3 and test a novel hypothesis that Rrm3 may prevent catastrophic re- replication of the genome.