Neisseria gonorrhoeae (Ng), a strict human pathogen, is the sole causative agent of gonorrhea. Gc possess a complex gene conversion system, the pilin antigenic variation system, (pilin Av), which alters the DNA sequence of the major pilin, PilE. Pilin Av results in the constant alteration of the surface exposed appendage, the pilus, allowing for evasion of immune memory. Pilin Av in Gc is one of many programmed recombination systems that allows us to study DNA recombination. Formation of an alternate DNA structure called a guanine quadruplex (G4) upstream of pilE, is required for pilin Av. This 16 base guanine-rich motif contains four tracts of guanines, with one or two loop thiamines. Transcription of a small noncoding-RNA is initiated within the motif on the cytosine rich strand and is required for pilin Av. I have found transcription of the G4 sRNA is rate limiting in Av and only 32 nucleotides of transcript is sufficient for Av. I developed a G4 chromatin immunoprecipitation assay to quantify G4 formation in the bacteria and found transcription of the sRNA is required for G4 formation. The sRNA forms a stable RNA:DNA hybrid (R-loop) as determined by R-loop chromatin immunoprecipitation with the C-rich strand of DNA. Using a regulatable RNase, which degrades the R-loops, I have found that extended R-loop formation is not required for G4 formation and pilin Av. Therefore, the sRNA functions to open the duplex, but once the G-rich DNA is single stranded, G4 formation and stability drives the initiation of pilin Av. Additionally, the G-C base-pairs within the G4 motif are required for pilin Av; however, the other requirements of this structure in pilin Av, such as loop size and composition, are unknown. Changing loop size and composition alters does alter pilin Av frequencies even though the G-C base pairs remain intact. RecA, a recombinase required for Av, binds the pilE G4 in vitro, so I compared how RecA interacts with different G4 structures. When comparing slight changes in G4 loop composition, I found the pilE G4 is the most stable structure. Therefore, stability and folding kinetics appear to be key in the pilin Av process. The G4 structure is stabilized by a cation, such as potassium, and here, I investigated whether intracellular potassium levels could regulate pilin Av. Finally, I hypothesize that the G4 and sRNA could contribute to a DNA nick or break during pilin Av. Therefore, I replaced the G4 with a yeast endonuclease, I-SceI, cut site to induce a nick or break at pilE, but did not detect any pilin Av. Understanding how the G4 and R-loop function in this recombination system will inform other biological systems which possess these alternate nucleotide structures.

Creator

• Prister, Lauren Leigh

DOI

• https://doi.org/10.21985/n2-6n3z-9x56

Subject

• Microbiology
• Genetics

Language

• en

Alternate Identifier

• etdadmin_upload_684526
• http://dissertations.umi.com/northwestern:14798

Keyword

• DNA recombination
• Antigenic variation
• Guanine Quadruplex

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright