gene regulation studies. 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. coli RNAPol makes frameshift errors when transcribing runs of As or Ts in the template DNA. My lab has demonstrated that the lambda N-Nus factor transcription antitermination complex modifies RNAPol and reduces these natural slippage events. N prevents transcriptional slippage, and since many intrinsic terminators have long U stretches, slippage and termination are likely to be interconnected processes. This is the first example of slippage being regulated, and it is possible that other N-like transcription regulators, e.g., HIV Tat, may also affect slippage. I study lambda to understand host functions with which the virus interacts, so that I can better understand their roles for the virus as well as the cell. These functions targeted by the virus are not only important for the virus but are also some of the most basic regulatory and sensory components of the cell. My work from the 1970's defined the post-transcriptional role of RNase III in retroregulation of lambda int gene expression from its 3'UTR. Several structures of RNase III have now been solved and contribute greatly to the understanding of the roles of Drosha and Dicer in RNAi 3'UTR gene regulation. We also described the role of RNase III in the processing of rRNA in coordination with the host Nus factors. Nus factors modify the RNAPol during rRNA transcription. The RNAPol-Nus transcription ensures rRNA folding, coordination of RNase III processing, and 30S ribosome assembly. Era is also intimately involved with 30S ribosome assembly. The Era-GTP complex binds to the 3'end of the maturing 16S rRNA, where it controls 16S processing, RNA folding, and the final stages of 30S subunit assembly. I have shown that mutants of Era block growth and cell division of E. coli, and I isolated a separation of function mutant that is competent for ribosome assembly and growth but is blocked for cell division. I propose that the Era-GTP/GDP cycle has check-points for growth and division, ensuring their coordination. Era homologues are conserved across all domains of life. Defects in human Era also cause dysfunction in the mitochondrial small ribosomal subunit, resulting in poor cell growth.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Haeusser, Daniel P; Hoashi, Marina; Weaver, Anna et al. (2014) The Kil peptide of bacteriophage ? blocks Escherichia coli cytokinesis via ZipA-dependent inhibition of FtsZ assembly. PLoS Genet 10:e1004217
Thomason, Lynn C; Sawitzke, James A; Li, Xintian et al. (2014) Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol 106:1.16.1-1.16.39
Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina et al. (2014) Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo. Proc Natl Acad Sci U S A 111:7308-12
Arbel-Goren, Rinat; Tal, Asaf; Friedlander, Tamar et al. (2013) Effects of post-transcriptional regulation on phenotypic noise in Escherichia coli. Nucleic Acids Res 41:4825-34
Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica et al. (2013) Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli. J Biol Chem 288:2700-10
Li, Xin-tian; Thomason, Lynn C; Sawitzke, James A et al. (2013) Bacterial DNA polymerases participate in oligonucleotide recombination. Mol Microbiol 88:906-20
Strader, Michael Brad; Hervey 4th, William Judson; Costantino, Nina et al. (2013) A coordinated proteomic approach for identifying proteins that interact with the E. coli ribosomal protein S12. J Proteome Res 12:1289-99
Thomason, Lynn C; Oppenheim, Amos B; Court, Donald L (2009) Modifying bacteriophage lambda with recombineering. Methods Mol Biol 501:239-51
Tu, Chao; Tropea, Joseph E; Austin, Brian P et al. (2009) Structural basis for binding of RNA and cofactor by a KsgA methyltransferase. Structure 17:374-85
Datta, Simanti; Costantino, Nina; Zhou, Xiaomei et al. (2008) Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages. Proc Natl Acad Sci U S A 105:1626-31

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