Molecular Mechanism Of Active Plasmid Partition

Stable transmission of duplicated genetic material to daughter cells is essential for all life forms. Active partitioning of low-copy-number plasmids has served as a simple model system for DNA segregation studies. Plasmid partition systems are composed of three elements:a cis-acting centromere-like region, a filament-forming motor protein ParA and a centromere-binding protein ParB. During plasmid segregation, multiple ParB proteins assemble on the centromere repeats to form a higher-order nucleoprotein complex, the partition complex. The partition complex further recruits motor proteins to form the segrosome. ATP-bound motor proteins can polymerize into filaments that separate attached plasmids to opposite poles of the dividing cell.Our lab previously identified a type Ib partition cassette in plasmid pCXC100 from Gram-positive bacterium Leifsonia. xyli subsp. cynodontis. In this plasmid, ParA is a typical Walker-type motor protein, but ParB contains no recognizable domain, raising the question whether ParB contains a novel DNA-binding domain and how it assembles with the centromere to form the partition complex. In this thesis, we have used structural and biochemical approaches to characterize the interaction of ParB with centromere DNA.(1) We have determined the structure of the C-terminal DNA-binding domain of ParB at 1.4 A resolution. The structure reveals a dimeric ribbon-helix-helix (RHH) fold, which is undetectable at the sequence level. RHH domains are classic DNA-binding domains and have been found in four other type Ib and typeⅡParB proteins. Our structure supports the prevalence of RHH domain in centromere binding. (2) We have extensively characterized the interaction of ParB with the centromere by hydroxyl radical footprinting and quantitative binding assays. The centromere core is composed of nine uninterrupted 9-nucleotide direct repeats with a consensus sequence AGNTGGAAA. The binding center of the ParB dimer is located between the fourth and fifth base pair of each repeat. We have shown that individual repeats are only moderately conserved and display 100-fold variation in affinity towards ParB (Kd=24-2900 nM). ParB binds to tandem repeats in a highly cooperative manner, facilitating the assembly of continuous partition complexes on centromeres containing degenerate binding sites. Our results reveal previously unrecognized similarity between the pCXC100 partition system and other type Ib systems with respect to ParB structure, centromere structure and cooperative ParB-centromere interaction.(3) We have determined a structure of ParB RHH domain bound to a nonspecific DNA sequence at 1.8 A resolution. In this structure, there are no interactions between bound ParB molecules, and the DNA adopts a straight and undistorted B-form conformation. The ParB-DNA interactions in the nonspecific-binding structure differ from those observed in the specific-binding structure. This structure is the first one in which a specific DNA-binding RHH domain interacts with a nonspecific DNA sequence and probably represents a state in which ParB searches the DNA prior to binding to the specific target sites.(4) We have determined a series of structures of ParB RHH domain in complex with tandem-repeat DNA, with the best resolution of 3.25 A. The two RHH domains bind to the adjacent repeats and interact with each other, providing the structural basis of binding cooperativity. The C-terminal tail of the RHH domain, which is otherwise missing in the free structure, inserts into the minor groove of a AT-rich DNA region. The C-terminal tail is protected from proteolytic digestion when ParB is in complex with tandem-repeat DNA, but not with single-repeat or noncognate DNA. The minor groove interaction has not been observed in other structures of RHH-DNA complex. The DNA duplexes stack end-to-end in the crystal and form a continuous helix, mimicking the whole centromere. The ParB RHH dimers wrap around the DNA helix, forming a superhelical higher-order structure. In collaboration with Tsinghua University, electron microscopy analysis of the negative-stained whole centromere DNA bound with ParB showed that the partition complex in solution also adopts a superhelical structure that has the same shape and dimensions as observed in the crystal. The partition complex structure of pCXC100 is distinct in terms of overall shape from those in type Ib pSM 19035 and typeⅡpSK41.