Mechanism of Bacterial SMC Complex MukBEF

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DESCRIPTION (provided by applicant): Chromatin structure undergoes marked, tightly controlled changes during the cell cycle. Maintaining proper chromatin structure is essential for faithful execution of such fundamental events as chromosome replication, recombination, gene expression or cell division. Errors in either of the processes often result in genomic instability and chromosomal alterations potentially leading to carcinogenesis or developmental defects. However molecular mechanisms underlying chromatin dynamics remain largely unknown. The SMC protein family is an emerging group of cellular ATPases responsible for large-scale chromatin reorganizations in organisms ranging from bacteria to humans. These proteins have been implicated in such diverse range of cellular functions as chromosome cohesion and condensation, DNA repair and dosage compensation. It was found recently that the mechanism of 13S condensin, a Xenopus SMC complex responsible for the chromosome condensation during cell division, involves direct ATP-dependent deformation of the large-scale DNA structure. This is an entirely novel kind of enzymatic activity. This project focuses on further mechanistic characterization of the SMC proteins using MukBEF, a bacterial analog of 13S condensin, as a model protein. The specific aims of this project are: (1) characterize biochemical activities of MukBEF; (2) investigate cell cycle regulation of MukBEF; (3) investigate the architecture of MukBEF-DNA complex using electron microscopy; (4) investigate DNA deformation by MukBEF using single DNA manipulation technique. These studies are expected to yield a comprehensive scheme of the MukBEF-catalyzed reaction. These data will contribute to our understanding of the intracellular functions of MukBEF and will illuminate the role of SMC proteins in large scale chromatin dynamics.