RECOMBINATION INTERMEDIATES: MECHANISMS IN RESOLUTION

点击翻译按钮获取中文摘要

This project is derived from our long term interest in the mechanisms by which genetic material is reassorted and rearranged. We propose to study the resolution (and the formation) of intermediates in one particular pathway of recombination. The system we are using, the Int-dependent pathway of bacteriophage lambda, is an example of what is called conservative transposition. The proposed experiments depend upon our recent finding that synthetic chi forms of att site DNA, which are formally analogous to Holliday-type recombination intermediates, are resolved by purified Int protein to specific predicted products. The specific aims of this proposal are: 1) To characterize the resolution of "synthetic" Holliday intermediates by purified Int protein, with special attention to the questions of specificity and directionality in resolution. 2) To determine how the resolution of Holliday structures is influenced by the accessory proteins IHF and Xis. 3) To determine how the resolution of Holliday structures is influenced by Int protein at sites distant from the region of strand exchange. 4) To study the relationships among the four Int protein binding sites in the Holliday crossover region and the role of DNA:DNA homology in this region. 5) To associate resolution of the Holliday intermediate with a specific domain of Int protein. 6) To find conditions or constructions that favor the isolation of "natural" Holliday intermediates.