Abstract The pathogenic Neisseria are obligate human pathogens that rely on several antigenic variation systems to colonize and cause disease in the human population. This proposal will further our studies into the molecular mechanisms for pilin antigenic variation in Neisseria gonorrhoeae. High-frequency gene conversion reactions mediate extensive changes in the pilin amino acid sequence with segmental recombination between one of 18 silent pilin copies and the single expressed pilin gene. Over the tenure of this grant, we have (1) identified most of the proteins involved in this process, (2) demonstrated that the pathogenic Neisseria carry diploid chromosomes that may facilitate gene conversion, (3) shown that the formation of an alternative DNA structure called a guanine quartet (G4) is necessary for pilin antigenic variation, (4) shown that transcription of a small RNA within the G4 is necessary for pilin antigenic variation, and (5) that the small RNA forms an RNA-DNA duplex (an R-loop). We will determine how new pilin variants replace the previous ones during pilin antigenic variation. We will test the hypothesis that the G4 structure and associated R-loop initiate gene conversion by altering chromosomal DNA replication. We will define how gene conversion occurs by applying various approaches to determine the steps between the initiation of the process and the creation of the final variant pilins by defining the molecules created and the effect of specific mutants on the generation of different intermediate molecules. Finally, we will test whether the N. gonorrhoeae diploid chromosomes are involved in gene conversion. The results of these cutting-edge studies will significantly impact the analysis of (1) Neisserial pathogenesis, (2) mechanisms of antigenic variation in Neisseria and other bacterial gene conversion mechanisms, and (4) the way programmed recombination is used in many cell types to alter gene expression or content. These studies will move all these fields forward and continue pilin antigenic variation as a model for diversity generation systems that use DNA recombination.