Molecular basis of bacterial chromosome segregation and organization

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PROJECT SUMMARY/ABSTRACT A fundamental problem in cell biology is understanding how DNAs are structured by compaction in the densely packed cellular environment, and accurately passed down to daughter cells. Chromosome-associated proteins are key factors in dynamically and accurately organizing chromosomes, and directly influence the replication, transcription, and translation of genetic information. As such, many diseases including various cancers are linked to malfunctioning of chromosome-associated proteins. In a majority of bacteria, ParABS partitioning system and structural maintenance of chromosomes (SMC) protein complex are main contributors for chromosome segregation and organization. The ParABS system is composed of ATPase variant ParA, short palindromic DNA sequence parS, and parS-binding protein ParB. The parS sites are located in the vicinity of bacterial origin of replication. A longstanding conundrum in the chromosome biology field is that ParB proteins are not only found on the parS sites but also associate extensive (10-20 kb) flanking regions – a phenomenon termed spreading. It had been attributed to the ability of ParB protein to bridge different segments of DNA, that allows long-distance interactions. A new way of thinking derived from recent discoveries that ParB protein is not merely a DNA binding protein but also a novel CTPase enzyme. It was proposed that cytidine triphosphate (CTP) binding to the ParS and its subsequent hydrolysis cycle drives self-loading of ParS onto parS sites and subsequent sliding away from the loading sites. However, this “clamp and sliding” model alone has limitations in accounting for in vivo chromosome immunoprecipitation data. Another critical role of ParB proteins is that they recruit SMC protein complex to the vicinity of the replication origin. However, little has been known about the SMC protein recruitment mechanism. Once recruited, bacterial SMC is thought to organize DNAs by actively extruding DNA loops. This simple mechanism that can explain many aspects of chromosome structuring is required to be demonstrated with bacterial SMC complex. The PI has almost 15 years of single-molecule techniques expertise and his lab is devoted to elucidating the mechanisms of various DNA-binding proteins and their impacts on chromosome structure. During the next five years, the PI’s laboratory will tackle the outstanding problems of underlying ParB and bacterial SMC working mechanisms and their interplays utilizing his single-molecule approaches and newly acquired surface plasmon resonance (SPR)-based expertise. Information one could extract from those proteins in traditional biology approaches is possibly averaged out due to the nature of simultaneous measurements of multiple proteins (ensemble measurements). Our approach will be expected to uncover hidden mechanisms with unprecedented details. The in vitro results will be corroborated by in vivo-based assays and theoretical modeling. The proposed work will pave the way for other future DNA-protein interaction studies. The long-term goal of the PI’s research program is to elucidate how different DNA-binding proteins and their cofactors cooperate to maintain the genome stability and dynamics.