Field of Science

What to propose to NIH?

How about this?

Goal: To fully characterize all of the biases and sequence specificities of transformational recombination in H. influenzae.

Specific questions to answer:
  1. 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?
  2. 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?
  3. 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?
  4. Is there any sequence specificity to the DNA degradation that accompanies uptake and translocation?
  5. What proteins interact directly with DNA during binding, uptake and translocation?
  6. What recombination biases affect indels? (How efficiently are different indels transfered by recombination?
  7. What are the recombination biases along the full length of the chromosome?
  8. How does mismatch repair affect the outcome of transformational recombination? How much of the recombination bias found by #7 is due to mismatch repair?

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