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Download or read book Understanding the Roles of the Mus81-Mms4 Structure-selective Endonuclease in the Reinitiation and Repair of Stalled Replication Forks in Saccharomyces Cerevisiae written by Erin Kimiko Schwartz and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The structure-selective endonuclease, Mus81-Mms4 (Eme1 in other eukaryotes), cleaves DNA joint molecules in the formation of meiotic crossovers and during reinitiation of stalled replication forks. The relevant substrate(s) on which the enzyme acts during these processes is a fundamental characteristic for the complex, and has been the subject of much debate in the field. In vitro biochemistry of the purified complex in budding yeast exhibits robust catalytic activity on nicked DNA junctions, whereas cleavage of intact, four-way Holliday junctions is inefficient and non-catalytic. Despite these findings, in vivo analysis of subunit association and hydrodynamic data of the purified complex suggest a larger oligomer, analogous to classic Holliday junction cleaving enzymes. In order to resolve the question of substrate specificity, we reevaluated the subunit architecture of the budding yeast complex in vitro and in vivo. The native molecular weight of the purified complex was determined by combining gel filtration and sedimentation coefficient data, and found to be consistent with a single heterodimer. These results were independently confirmed by single particle analysis of the purified protein complex using gradient fixation. Complementary co-immunoprecipitation experiments confirmed these findings in vivo for both soluble and chromatin-bound complexes, and in the presence and absence of DNA damage-induced phosphorylation. These results can be combined in complete agreement with the previously published biochemistry results to form a consistent model where a single heterodimer, with a single active site, processes nicked DNA substrate(s) for effective DNA repair. In addition to addressing the question of substrate specificity, we evaluate the functional context of Mus81-Mms4 activity. In budding yeast, we identify a novel role for Mus81 in telomere stability. In the absence of telomerase, Mus81 is required for viability during telomere shortening and is not involved in recombination-mediated telomere lengthening, as has been proposed for human MUS81. This requirement for Mus81 becomes even more important in the presence of replication fork stalling agents, suggesting that the enzyme is supporting replication through telomere DNA during recombination-mediated telomere lengthening. We further correlate Mus81 post-translational modification to protein stability and function, as well as identify novel interactions between Mus81-Mms4 and components of the INO80 complex. These results can act as the foundation for new studies on the function and context of Mus81-Mms4 activity.