ZIA BC 010336 (ZIA) | |||
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Title | Lambda Genetic Networks and Lambda Red-Mediated Recombination | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Court, Donald | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $1,316,136 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Bioengineering (15.0%) Cancer (100.0%) Gene Therapy (25.0%) |
N/A | ||
Research Type | |||
Systemic Therapies - Discovery and Development Application of Model Systems |
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Abstract | |||
Recent characterizations of the lambda genetic network have provided a framework for systems biology approaches using lambda as a prototype for theoretical modeling methodologies, which have become important for addressing signal transduction, cancer development and other complex genetic networks of eukaryotes. Co-evolution of lambda with E. coli has produced genetic systems that are exquisitely connected to the host's most basic functions. By examining the interface between lambda and host systems, my lab follows the trail of the phage to understand what is most important and vital to both cellular life and viral exploitation of cellular systems. The virus provides clues as to how those cellular functions work and how to study them. All of the work in my lab has derived from this philosophy. New discoveries change our perception of the lambda genetic network and affect models describing it. Two genes, rexA and rexB, cotranscribed with the cI repressor gene, have been largely ignored for contributions to the complex lambda genetic network controlling repressor activity and its synthesis. We found a new role for RexA in this regard as it appears to interact with CI repressor to promote induction. We also have evidence suggesting that the immunity terminator overlaps the end of the rexB gene, and that translation of RexB modulates the terminator, affecting transcription levels of the cI gene. Thus, the classic Rex exclusion system is intimately involved with lambda immunity control, adding further subtlety to the bistable genetic switch model of lambda that has provided a basis for mathematical models of gene regulation. We have developed the lambda homologous recombination functions Red as reagents for recombineering, a revolutionary in vivo genetic engineering technology that has enabled new approaches for functional genomic studies from bacteria to man. Recombineering allows modification of genomic clones from any organism, and is being used for developing model systems for cancer and other disease-related research. Similar recombineering systems are being developed in other bacteria, including pathogens, and can be used to develop vaccines, molecular targets for antibiotics, phage therapy, and biodefense. We demonstrated that short single-strand oligonucleotides recombine with homologous targets on replisomes in E. coli and other bacteria. Phage recombinases, like Beta and RecT, stimulate this oligo recombination above low endogenous levels in the cell. Results suggest that the molecular mechanism for initiation of oligo recombination by the two recombinases Red Beta and RecT differ. Beta requires replication of the target DNA to initiate and generate a recombination intermediate, whereas, RecT does not require DNA replication to generate an intermediate. This supports the premise that Beta acts by ss-strand annealing at the replication fork, whereas RecT forms D-loops by strand invasion. We plan to similarly characterize initiation of recombination by other recombinases including HSV-1 ICP8. Faithful transcription of DNA is dependent on RNA polymerase (RNAPol) maintaining accuracy in matching the incoming nucleotide to the template to prevent misincorporation errors and maintaining the register between the template and the transcript to prevent slippage errors. Failure to faithfully transcribe the template has been suggested to lead to a variety of diseases including certain cancers, Down's syndrome, and Alzheimer's disease. My lab demonstrated that the RpoC D1143P polymerase misincorporation mutant caused genetic instablility of an IS2 insertion element. Instability was enhanced further when combined with defects in the GreA and GreB transcription factors. I speculate that RNAPol complexes arrest after misincorporation and interfere with DNA replication. This could lead to DNA repair with the potential for rearrangements, a common cause of cancers in higher organisms. Another type of mistake is transcriptional slippage. E. |