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Current Research

Recent mechanistic studies

DNA replication is essential in all free-living organisms, and is strictly coordinated with cell growth. Our ability to manipulate this process to promote or block cell growth, as in the case of infection or cancer, requires a mechanistic understanding that includes the function of each individual replication protein.

The focus of the Kaguni laboratory is to understand the biochemistry of replication initiation, and how this process is regulated to ensure that it occurs only once per cell cycle. One mechanism of regulation is through Hda and the dimeric b clamp, which stimulate the hydrolysis of ATP bound to DnaA. The resulting DnaA-ADP is not active in replication initiation. In recent collaborative work with two other labs, we reported on the crystal and solution structure of the Hda-β clamp complex that contains two pairs of Hda dimers sandwiched between two β clamp rings. Among the three interacting surfaces between Hda and the β clamp, and between Hda protomers, the interface between Hda monomers occludes a critical arginine residue that is involved in the hydrolysis of ATP bound to DnaA. Hence, Hda in the octameric complex is unable to regulate the activity of DnaA. This study indicates that the octameric complex must disassemble to permit the arginine residue of Hda to interact with DnaA.

At a DNA replication origin, helicase loading often requires the dynamic interactions between the DNA helicase, a ring-shaped enzyme, and a companion protein. In Escherichia coli, the DNA helicase is DnaB and DnaC is its loading partner. In a recent studies, we addressed the importance of DnaB-DnaC complex formation as a prerequisite for helicase loading. Our results show that the DnaB ring naturally opens and closes, and that specific amino acids near the N-terminus of DnaC interact with a site in DnaB’s C-terminal domain to trap it as an open ring. Evidence indicates that DnaC alters the helical hairpins in the N-terminal domain of DnaB to occlude this region from interacting with primase. Other observations suggest that DnaC and primase have opposing effects on each other. Apparently, the binding of DnaC or primase to the respective domains of DnaB transduces a conformational change to the other domain to interfere with binding of the second protein. On the basis of the dynamic interactions of DnaC and primase with DnaB, we suggest the novel idea that DnaC controls the access of DnaB to primase.

Separate work focused on the binding of DnaC to single-stranded DNA, which was speculated to be required for DnaC to function in DNA replication. Genetic and biochemical characterization of mutant proteins designed to be defective in DNA binding revealed an unexpected increase in affinity for DNA. Apparently, the gain-of-function mutations cause DnaC to become defective in DNA replication. We suggest that the impaired ability to dissociate from the single-stranded DNA causes DnaC to remain bound to DnaB, which blocks its activation as a DNA helicase.

Planned mechanistic experiments

In bacteria, one of the critical functions of DnaA is to load the replicative helicase at the replication origin, but the biochemistry of this process is very unclear. The high sequence conservation of DnaA homologues together with biochemical studies support the idea of a similar mechanism of replication initiation among bacteria. A major objective is to obtain new insight into helicase loading by determining the cryo-EM structure of a replication intermediate named the ABC complex that contains Escherichia coli DnaA, DnaB and DnaC assembled at a DnaA box-containing hairpin. We suggest that this structure is comparable to one that leads to formation of the replisome that moves leftward on the bacterial chromosome. Moreover, this aim will lead to important new insight on the relative positions of DnaA, DnaB and DnaC in the complex, which may be extrapolated to possible structures and orientations of the proteins at the replication origin of bacteria. To validate the cryo-EM structure, we will perform hydrogen/deuterium exchange analysis of the ABC complex to identify the interacting sites of proteins in the macromolecular structure, and will also confirm the structure via biochemical and genetic methods.

 

Development of novel antibiotics

In free-living organisms, the enzymes that function at the respective stages of DNA replication differ in amino acid sequence, and their three-dimensional structures. Among bacteria, protein homologues are highly conserved. Hence, compounds that target this essential pathway should be specific to bacteria. A long-term aim is to identify compounds that inhibit DNA replication in bacteria with the goal of developing them into novel antibiotics. Much attention has been given to the transfer of multi-drug resistant bacteria from food animals to humans, and the threat of such bacteria to human health. Acknowledging the danger, the World Health Organization in 2017 issued a tiered list of multidrug-resistant pathogens. However, infectious diseases also imperil food animals. Using the example of bovine respiratory disease, it is the most common and costly disease in the U.S. beef cattle industry. The emergence of pathogens resistant to drugs used to treat the respiratory disease has increased in feedlot cattle. Thus, new antibiotics that are effective against multi-drug resistant bacterial are critical to both human health and the food animal industry.

Recently, Kaguni’s lab established and optimized a high-throughput assay system that requires almost all of the same proteins required for duplication of the E. coli chromosome. Using this assay, we identified compounds that specifically inhibit DNA replication. These compounds will be studied further.

Future work on the development of novel antibiotics

The preliminary studies on the development of novel antibiotics are the foundation for three objectives. One is to identify the proteins inhibited by the respective compounds via biochemical approaches and to confirm the results by genetic methods. A second is to determine the binding site of a compound by molecular dynamics methods in combination with mutational analysis of the target protein. A third objective is to construct chemical derivatives in a progressive process in which the successive derivatives are designed to bind with greater affinity and may be more potent. Their biochemical characterization will lead to the determination of the molecular mechanisms of inhibition. The work may lead to new antibacterial agents that are effective in preserving animal and human health.