This proposal addresses two different aspects of chromosome behavior in bacteria: the role of DNA adenine (dam) methylation in regulating DNA replication and the bacterial cell cycle and the nature of transposition by transposon Tn10 and its component IS10 sequences. I. Dam methylation plays a critical role at the E.coli origin of replication. Immediately after replication initiation, the hemimethylated origin is sequestered for about 15 minutes and then released. This sequestration is thought to play a role in preventing secondary replication initiations and may also play a role in chromosome segregation. The promoter for the dnaA gene, which maps a minute away from the origin, is similarly sequestered; this sequestration may play a role in preventing secondary initiations and/or be part of a cell-wide signal that replication has initiated. We propose to investigate the nature of sequestration at oriC and dnaA, the nature of the release from sequestration, and the role of sequestration at dnaA. To these ends, we propose to isolate three types of bacterial mutations which affect this process, to determine the cis-dominant determinants required for sequestration at dnaA, to investigate the effects of varying the level of dam methylase on sequestration and secondary replication initiations, and to investigate the biological consequence of moving the dnaA gene away from oriC. II. Transposable elements play a major role in mediating DNA rearrangements within and between prokaryotic organisms. We propose to investigate the nature of the TN10/IS10 transposition reaction through a combination of genetic, biochemical and physical approaches. A. Mechanistic analysis in vitro and in vivo. We propose to define the requirements for intermolecular transposition, determine the nature of and functional dependence among strand cleavages and ligations, investigate the special role of terminal transposon basepairs, continue our analysis of insertion specificity, investigate the mechanistic basis of transposition length dependence, and investigate processing of gapped donor molecules. B. Structure and function of IS10 transposase: physical approaches. We propose to characterize in detail two types of stable protein/DNA complexes that we have identified as intermediates in the transposition reaction. Analysis will include definition of important DNA contacts by interference and footprinting assays, determination of protein/DNA stoichiometry, examination of protein structure/function relationships by DNA/protein crosslinking, and investigation of the topology of synaptic complex formation. We also propose to search for less stable transposase/DNA complexes that are likely to exist but have not yet been identified. C. Structure and function of IS10 transposase: genetic approaches. We will continue to analyze phenotypically and biochemically three types of mutants already isolated (SOS+Tnsp, ATS, and REM), to isolate intragenic revertants of mutants of the first two types, and to search for new types of transposase mutants.
Showing the most recent 10 out of 53 publications
