Faithful chromosome replication is a central mission of every dividing cell. Although the machinery for DNA replication is among the most conserved across the tree of life, the regions of chromosomes at which replication initiates 'origins of replication' vary widely across species both in their structure and in their locations. Chromosome replication has been best studied in the model organism baker's yeast. Replication origins in yeast are defined by short stretches of DNA sequence, with ten to fifty origins scattered across each chromosome. In contrast, human chromosomes have many hundreds to thousands of origins per chromosome unspecified by DNA sequences. Despite these striking differences in origin structures, little is known about how origins of replication evolve, and how they may relate to chromosomal rearrangements that take place during evolution. Baker's yeast and its other budding yeast relatives offer an unparalleled advantage in addressing these gaps in our knowledge: many strains and species have been sequenced and are amenable to experiments first performed in baker's yeast. This research will capitalize on the suite of chromosome-wide assays to discover yeast replication origins, delineate their essential sequences, and measure the regulation at each site in its native chromosomal context. Moreover, the opportunity to compare multiple yeast species that had undergone a whole genome duplication (WGD) with other species that hadn't experienced the event can provide unique insights into the forces shaping replication and chromosome organization in the face of a massive genome upheaval. The work will focus on how origins evolved over a 500 million year timescale. The aims are grounded in the context of testing the following broad hypotheses: First, post-ÂWGD species will show greater diversity of origin sequences and locations than pre-ÂWGD species due to relaxation of selection during genome loss following the WGD event. Second, in contrast, closely related species will share common origin sequences but not chromosomal locations. Finally, changes that occur to the protein machinery that recognizes origins are responsible for driving the diversification of the DNA sequences of origins. Given the increasingly appreciated role of origins in maintenance of chromosome integrity, the results will add an important missing element to understand the evolution of chromosome structure.
Broader Impacts: This work will have broad impacts in undergraduate and public science education, inclusion of underrepresented groups, and scientific understanding. A new partnership with Shoreline Community College has been proposed specifically for this project and will, in particular, result in access to research and student-Âdriven, inquiry-Âbased learning for a wider cross-Âsection of students than would otherwise be possible. In particular, the intern experience will build confidence and prepare students for jobs as technicians in university or biotech research labs, and may spark their interest in pursuing further education. All three principal investigators have strong records of educating undergraduates, including women, underrepresented minorities, and students from primarily undergraduate institutions. The methods and molecular resources developed will be of interest to the larger scientific community, as has been the case for other techniques previously developed by these principal investigators. Furthermore, contributing to the understanding of evolutionary processes is a benefit to science and society. Specifically, rapid evolution of a highly conserved process is an under-Âstudied phenomenon, and the results are likely to shed light on the natural variation found in other essential cellular processes.
