How bacterial SMC complexes organize chromosomes

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Project Summary The organization and segregation of replicated chromosomes are fundamental to living systems. Structural maintenance of chromosomes (SMC) complexes play central roles in these processes in all domains of life. These ring-shaped ATPases share common structures and inter-subunit contacts, consistent with a common mechanism of action. Recent studies in Bacillus subtilis and eukaryotes have provided compelling in vivo and in vitro evidence that SMC complexes utilize ATP hydrolysis to extrude DNA loops. However, it is unclear how SMC complexes extrude DNA loops when the chromosome is coated by numerous proteins and acted upon by replication and transcription machineries. My research group aims to determine how SMC complexes act when they encounter the replisome or other condensin molecules. We will also characterize how SMC complexes are released from the chromosome when they reach the terminus region. Finally, we will explore SMC’s role in the organization and dynamics of a multipartite bacterial genome that contains both a circular and a linear chromosome. Taken together, our work has the potential to provide the general principles of chromosome folding and compaction in all organisms. In this proposed application, I am requesting an administrative supplement to support an undergraduate in my lab, Ms. Lindsey Way, to do a research project in my lab during the summer of 2023. The goal of this project is to support Ms. Way for full-time summer research experience in bacterial cell biology and molecular biology. Ms. Way is currently a Junior at IU. She is determined to pursue a Ph.D. in biomedical sciences. A full- time research in this summer will better prepare her for graduate school. Ms. Way’s project will investigate the interactions between SMC complexes and a collection of histone-like proteins from bacteria and archaea, including HupA and H-NS from E. coli, and HMfA and HMfB from Methanothermus fervidus. Ms. Way will: 1) build plasmids and strains to express these proteins fused to mCherry; 2) visualize these fusion proteins using fluorescence microscopy; 3) quantify their expression using Western blotting; 4) monitor the consequences of the SMC-histone interactions by genome-wide chromosome conformation capture Hi-C assays. Ms. Way will present in our weekly lab meetings. She will also present a poster in the Indiana University Microbiology Annual Retreat in October 2023.