Molecular and genetic analysis of ORC target site selection and origin function in Saccharomyces cerevisiae

The origin recognition complex (ORC) plays a pivotal role in genome duplication by binding to the specific positions on chromosomes that will serve as DNA replication origins. Although ORC is conserved in all eukaryotes, the DNA sequence elements that specify origins and ORC binding are not. In the budding yeast Saccharomyces cerevisiae ORC binds to specific DNA elements within origins. However, in higher eukaryotes ORC shows no DNA sequence specificity and ORC-origin interactions are less well defined and rely more on DNA and chromatin structure instead. These differences raise the question of whether ORC target site selection mechanisms are conserved between yeast and higher eukaryotes. Focusing on conserved features of ORC and examining their roles in ORC target site selection can provide insights into conserved features of origins needed to facilitate ORC binding.In this study, I focused on the conserved N-terminal bromo adjacent homology domain of Orc1 (Orc1BAH) in yeast DNA replication. Recent studies reveal that BAH domains are nucleosome binding modules, but the function of the Orc1BAH domain has not been addressed in yeast. Biochemical experiments reveal that the Orc1BAH domain is necessary for ORC's stable association with chromatin and genome wide analyses furthermore show that the Orc1BAH domain is necessary for ORC's full association with the majority of yeast origins. Two distinct classes of origins can be identified: those that are highly dependent on the Orc1BAH domain (orc1bahDelta-sensitive) and those that do not require this domain for ORC's full association (orc1bahDelta-resistant). Genetic analyses provide evidence that the Orc1BAH domain is physiologically relevant to DNA replication in vivo. Molecular and bioinformatic comparisons of the two classes of origins reveal that they differ in terms of their ORC binding sites and surrounding chromatin structure. These data support a model in which yeast ORC selects origins through a combination of ORC-DNA and ORC-chromatin interactions and suggest that ORC-chromatin interactions are a conserved feature of ORC's selection of origins within eukaryotic chromosomes.