Since the discovery of the double-helical structure of DNA 60 years ago, a wealth of kinetic and structural data have illuminated many of the mechanisms DNA polymerases use to achieve efficient and accurate DNA replication. Even so, our understanding is far from complete, especially since many different polymerases are now known to be required for complete genome duplication. Here, we focus our research on two types of polymerases from the bacterial pathogen Staphylococcus aureus: the essential C-family replicative polymerases and the Y-family translesion polymerases. The C-family polymerases include the highly accurate PolC, which is unable to efficiently copy damaged DNA, and the error-prone DnaE, which initiates DNA synthesis from RNA primers. The Y-family polymerases are able to replicate damaged DNA but are highly error-prone. We will use kinetic, structural and molecular genetic techniques to determine how these polymerases replicate the bacterial genome with high accuracy overall, but also create mutations that give rise to antibiotic resistance. Since DNA replication is such a fundamental process, the research will have broad implications for other bacteria.
The proposed work will provide a detailed structural and kinetic understanding of the mechanisms of DNA replication in Gram-positive bacteria, establishing a strong foundation for combating antibiotic resistance and developing new antibacterial approaches. The research focuses on the essential polymerases and the translesion polymerases from the low GC-content Gram-positive bacterium Staphylococcus aureus. The MRSA strains of S. aureus are major sources of hospital-associated infections and are of concern because they are increasingly resistant to antibiotics.
