Contribution of Cis-acting Elements and Trans-acting Host Factors in DGR-Mediated Mutagenic Homing

Author(s): Czornyj, Elizabeth | Advisor(s): Miller, Jeff F | Abstract: Diversity-generating retroelements (DGRs) are family of retroelements that introduce nucleotide variability within defined protein-encoding DNA sequences. Sequence variation is site specific and occurs through a unique reverse transcriptase mediated process called mutagenic homing. DGRs were originally identified in the Bordetella phage BPP-1 and have since been identified in plasmids, bacteriophage and bacterial genomes. Moreover, DGRs were recently identified in Archaea and their viruses. Although DGRs are wide spread in nature and protein diversification has been demonstrated in both phage and bacterial systems, the precise mechanism of DGR mutagenic homing remains to be elucidated. We have demonstrated that mutagenic homing requires specific nucleotide sequence and structural elements, including target site recognition sequences, which include a DNA stem-loop/cruciform structure. We recently demonstrated that in addition to base pairing interactions in the stem, the specific sequence and length of the 4nt loop are critical for DGR function. In vitro and in vivo analyses of the stem-loop structure indicate that the loop nucleotide composition has a major effect on stem-loop/cruciform formation and stability, and is thus critical for DGR function. In addition to influencing structure stability, we demonstrate that the orientation of the loop nucleotide sequence determines target site recognition during mutagenic homing. Stem-loops have been identified in most DGRs and our analysis of similar elements from disparate species indicates that these conserved elements are functionally interchangeable and fundamental to target site recognition. We propose that the stem-loop/cruciform structure serves as a recognition element for DNA processing events that culminate in cDNA synthesis, diversification, then integration. Since bioinformatic and functional analysis of DGRs reveals the lack of sequences predicted to encode enzymes involved in DNA or RNA processing events, we postulated host-encoded trans-acting factors play a pivotal role in mutagenic homing. To identify host-encoded factors that directly or indirectly influence the Bordetella phage BPP-1 DGR homing, a random transposon-insertion library was created. Individual transposon mutants were screened for insertions that had a significant effect on DGR homing as measured by a quantitative Km resistance assay. As an alternative approach to identify host factors, we performed targeted mutagenesis of candidate genes involved in DNA- and RNA-processing activities, including but not limited to ssDNA-specific exonuclease, ATP-dependent DNA helicase, RNase H, RNase E, and other putative endoribonucleases. Mutants were screened for their ability to support mutagenic homing using phage tropism switching assays and we identified a subset of mutations in genes encoding DNA- and RNA-processing enzymes that decreased tropism switching. Taken together, our findings indicate that mutagenic homing involves both DGR-encoded and host-encoded factors that play a role in the diversification of target proteins, providing insight into a highly conserved mechanism for DNA editing.