Structure and Function of the NES Relaxase and C-terminal Domains Required for Vancomycin Resistance Transfer

Antibiotic resistance has become a large burden in the healthcare setting due to increasing rates of mortality and increased healthcare costs due to extended treatment. Staphylococcus aureus is one of the most prevalent healthcare associated infections (HAI) and has recently become resistant to vancomycin (VRSA). This is a serious problem as vancomycin is one of the last lines of defense against resistant gram-positive bacteria. Given this new resistance combined with the fact that novel antibiotic approval has steadily been decreasing we sought to better understand the process of conjugative plasmid transfer (CPT) for the vancomycin resistance plasmid in S. aureus, pLW1043. We focused on a key enzyme involved in CPT, the relaxase. The relaxase on pLW1043 has been identified through sequence analysis and is termed the nicking enzyme of S. aureus (NES). The relaxase initiates and terminates CPT. When CPT has been initiated the relaxase will bind to and nic the DNA at a specific location on the plasmid termed the oriT. We hypothesized that inhibition of the relaxase would halt the spread of antibiotic resistance and could result in a novel class of antibiotics. To investigate this hypothesis, we set out to determine the structure of NES, understand its biological function, and investigate its interaction with the oriT DNA. Little work has been conducted in gram-positive bacteria and by understanding this critical enzymes role in CPT we hoped to provide new insights to the scientific community. In the following chapters we discuss the structure of the N-terminal relaxase domain in complex with DNA. Furthermore, we will use this structural information combined with biochemical assays to identify how NES recognizes and site specifically cleaves the DNA at the proper location. We will then investigate the activity of NES with different metals and shed light on the elements required for proper cleavage and religation. Using this data, we then propose a novel strategy for the inhibition of NES and the development of novel antibiotics. Finally, we will analyze the structure of the C-terminal domain, which has no known function, and propose a role for its function in the DNA processing reaction