Recognition of oriT at the termination of conjugal transfer by MobA, the R1162 DNA strand transferase

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dc.contributor.advisorMeyer, Richard John, Ph. D.en
dc.creatorBecker, Eric Christianen
dc.date.accessioned2011-04-14T17:39:20Zen
dc.date.available2011-04-14T17:39:20Zen
dc.date.issued2002-05en
dc.descriptiontexten
dc.description.abstractR1162 is efficiently mobilized during conjugation by IncP-1 plasmids such as RK2 and R751. Transfer is terminated when the transferred strand, linearized at the 38 base-pair origin of transfer (oriT), is recircularized by the plasmid encoded protein MobA. For strand rejoining, MobA covalently linked to the 5' end of the strand rejoins the ends by a reversible transesterification reaction. The minimal oriT fragment of R1162 contains a highly conserved 12 base region (core) including the cleavage site and a ten base imperfect inverted repeat (IR) that is not highly conserved. From those oligonucleotides with a partially degenerate oriT core base sequence, the subpopulations that are bound by MobA, cleaved and rejoined by this protein and support termination of transfer were identified. Both the IR and the adjacent core bases TAA, are needed for tight binding to MobA, whereas the location of the dinucleotide YG determines the site of strand cleavage. At the IR MobA stabilizes duplex DNA during gel electrophoresis and binds weakly to oligonucleotides lacking the outer arm of the inverted repeat, supporting a model where secondary structure at IR provides a duplex region needed for binding during termination (Zhang and Meyer 1995). Significantly altered IR sequences did allow strong binding to MobA yet completely different IR sequences did not, indicating the IR serves a structural role for binding with low base specificity. A 184 residue aminoterminal MobA fragment capable of binding and cleaving oriT was identified by using phage display and partial enzymatic digestion of the protein. No smaller fragments that could bind or cleave oriT were identified. An active nucleoprotein intermediate consisting of MobA covalently linked to the 5' end of the cleaved oriT was used to show that a single molecule of MobA is sufficient to carry out all the DNA processing steps during transfer.en
dc.description.departmentMicrobiologyen
dc.format.mediumelectronicen
dc.identifier.urihttp://hdl.handle.net/2152/10920en
dc.language.isoengen
dc.rightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.en
dc.rights.holderRestricteden
dc.subjectGenetic transformationen
dc.subjectConjugation (Biology)en
dc.subjectPlasmidsen
dc.titleRecognition of oriT at the termination of conjugal transfer by MobA, the R1162 DNA strand transferaseen
thesis.degree.departmentMicrobiologyen
thesis.degree.disciplineMicrobiologyen
thesis.degree.grantorThe University of Texas at Austinen
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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