Goal: To fully characterize all of the biases and sequence specificities of transformational recombination in H. influenzae.
Specific questions to answer:
- Is DNA binding a distinct step that precedes the initiation of DNA uptake? If so, does it have the same sequence specificity as DNA uptake? Does it have any topological specificity?
- What is the complete sequence specificity of DNA uptake? How absolute is the requirement for a good match to the USS consensus at uptake (are non-USS DNAs taken up at lower frequency or not at all)? This will be investigated with plasmids or short fragments containing 12% degenerate USSs, and with ones containing completely random sequences (we could create these or just use fragments of unrelated DNA). Does uptake have any topological specificity?
- Does the translocation step impose any sequence specificity? How strict is the requirement for a pre-existing free end (does circular DNA sometimes get cut or nicked in the periplasm, or transported intact into the cytoplasm?
- Is there any sequence specificity to the DNA degradation that accompanies uptake and translocation?
- What proteins interact directly with DNA during binding, uptake and translocation?
- What recombination biases affect indels? (How efficiently are different indels transfered by recombination?
- What are the recombination biases along the full length of the chromosome?
- How does mismatch repair affect the outcome of transformational recombination? How much of the recombination bias found by #7 is due to mismatch repair?