UC Berkeley

Primases are DNA-dependent RNA polymerases found in all cellular organisms. In bacteria, primer synthesis is carried out by DnaG, an essential enzyme that serves as a key component of DNA replication initiation, progression, and restart. A long-standing question in the field of polymerase biochemistry is how the striking structural differences between DnaG and other single-subunit polymerases translate to differences in function. A specific barrier to addressing how DnaG relates to other polymerases is a relatively poor understanding of how DnaG associates with its own substrates. We investigated this issue through a multipronged approach. First, we have examined one of the earliest stages in primer synthesis by solving crystal structures of the S. aureus DnaG catalytic core bound to metal ion cofactors and either individual nucleoside triphosphates. Second, we investigated how the known classes of primase inhibitors, including two naturally-prevalent inhibitors, the nucleotidyl alarmones, pppGpp and ppGpp, and 2´-deoxyribonucleoside triphosphates, control primer synthesis. When taken together with both biochemical analyses and comparative studies of enzymes that use the same catalytic fold as DnaG, these studies pinpoint the predominant nucleotide-binding site of DnaG, leading to a reconciled model for primer synthesis by DnaG. Our studies of the inhibitors of DnaG illustrate how the induction of the stringent response in bacteria interferes with primer synthesis, and explain the ability of DnaG to differentially discriminate between dNTPs and rNTPs depending on the stage of primer synthesis. Finally, many new tools were developed, which combined with our improved understanding of how DnaG functions in substrate recognition and catalysis could motivate renewed interest in the research and development of DnaG-targeted antimicrobial agents.