Phage Mu transposes by two distinct pathways depending on the specific stage of its life cycle. A common θ strand transfer intermediate is resolved differentially in the two pathways. During lytic growth, the θ intermediate is resolved by replication of Mu initiated within the flanking target DNA; during integration of infecting Mu, it is resolved without replication, by removal and repair of DNA from a previous host that is still attached to the ends of the incoming Mu genome. Our studies show that the cryptic endonuclease activity reported for the isolated C-terminal domain of the transposase MuA, which is not observed in the full-length protein or in the assembled transpososome in vitro, is required in vivo for removal of the attached host DNA or “5’flap” after the infecting Mu genome has integrated into the E. coli chromosome. I have identified additional phage and host factors required for flap removal in vivo, which include an early Mu protein called Ner, and the E. coli protein ClpX. Ner regulates bidirectional transcription through the Mu transposition enhancer, while ClpX, a molecular chaperone, is known to interact with the C-terminus of MuA to remodel the transpososome for replication. The transpososome is a multi-subunit MuA complex assembled on the two paired ends of Mu. The enhancer DNA segment serves as an essential scaffold for transpososome assembly, and remains stably associated with θ strand transfer MuA complexes. I hypothesize that Ner-regulated transcription through the enhancer remodels transpososome conformation in the presence of ClpX, promoting activation of the MuA endonuclease, which resects flanking DNA during the repair pathway of Mu transposition. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/ETD-UT-2010-08-1815 |
| Date | 14 December 2010 |
| Creators | Choi, Wonyoung |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
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