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ItemManipulating Cells: Innovative Research at Georgia Tech(Georgia Institute of Technology, 2012-10-18) Doyle, Donald ; Storici, Francesca ; Newman, Jonathan P.[Doyle] Nuclear hormone receptors control the expression of genes in response to small molecule hormones. In performing this activity, the receptors must specifically recognize small molecules, DNA, and other proteins. The amino acids that recognize each of these substrates are varied using genetic engineering techniques until a receptor with novel recognition is created. The original and new receptors are studied using a variety of biophysical techniques to elucidate the principles behind the new activity. This exercise provides both new knowledge for future protein engineering and real materials for research and medical applications.
ItemAAV recombineering with single strand oligonucleotides(Georgia Institute of Technology, 2009-11) Hirsch, Matthew L. ; Storici, Francesca ; Li, Chengwen ; Choi, Vivian W. ; Samulski, R. JudeAdeno-associated virus (AAV) transduction initiates a signaling cascade that culminates in a transient DNA damage response. During this time, host DNA repair proteins convert the linear single-strand AAV genomes to double-strand circular monomers and concatemers in processes stimulated by the AAV inverted terminal repeats (ITRs). As the orientation of AAV genome concatemerization appears unbiased, the likelihood of concatemerization in a desired orientation is low (less than 1 in 6). Using a novel recombineering method, Oligo-Assisted AAV Genome Recombination (OAGR), this work demonstrates the ability to direct concatemerization specifically to a desired orientation in human cells. This was achieved by a singlestrand DNA oligonucleotide (oligo) displaying homology to distinct AAV genomes capable of forming an intermolecular bridge for recombination. This DNA repair process results in concatemers with genomic junctions corresponding to the sequence of oligo homology. Furthermore, OAGR was restricted to single-strand, not duplexed, AAV genomes suggestive of replication-dependent recombination. Consistent with this process, OAGR demonstrated oligo polarity biases in all tested configurations except when a portion of the oligo targeted the ITR. This approach, in addition to being useful for the elucidation of intermolecular homologous recombination, may find eventual relevance for AAV mediated large gene therapy.
ItemMechanisms of DNA Repair and Gene Targeting with DNA and RNA Molecules(Georgia Institute of Technology, 2009-06-06) Storici, Francesca
ItemHypermutability of Damaged Single-Strand DNA Formed at Double-Strand Breaks and Uncapped Telomeres in Yeast Saccharomyces cerevisiae(Georgia Institute of Technology, 2008-11) Yang, Yong ; Sterling, Joan ; Storici, Francesca ; Resnick, Michael A. ; Gordenin, Dmitry A.The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase f. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UVinduced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution.