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Michael Seidman, Ph.D., Senior Investigator Chief, Section on Gene Targeting Laboratory of Molecular Gerontology Phone: 410-558-8565 Fax: 410-558-8157 E-mail: seidmanm@grc.nia.nih.gov |
| Biography: Dr. Michael Seidman received his Ph.D. in biochemistry from the University of California, Berkeley and did his postdoctoral work at the NIH and Princeton University. He worked at the National Cancer Institute where he and his colleagues developed the supF reporter plasmids for studies of mutagenesis in mammalian cells. He held executive positions in the biotechnology industry before coming to NIA. |
| Gene Targeting: We are interested in the metabolism of site specific DNA damage in mammalian genomes, with a current focus on DNA crosslinks. These form in DNA following reaction with lipid peroxidation products generated during normal cellular metabolism, and also as a result of treatment with various chemotherapeutics. Despite many years of effort crosslink repair in mammalian cells is not well understood, in part because of the multiplicity of gene functions and pathway options involved. We have taken a novel approach to this problem by following the fate of crosslinks introduced at a specific site in the genome of mammalian cells. In order to do this we have developed gene targeting reagents based on triple helix forming oligonucleotides (TFOs). The oligonucleotides carry chemical modifications that allow them to form sequence specific triplexes with genomic DNA target sequences under physiological conditions. We have linked psoralen, a DNA crosslinker, to the oligonucleotides and have used the conjugates to introduce crosslinks at a specific chromosomal target site in living cells. Efficient targeting requires not only effective oligonucleotides, but also manipulation of the cell biology to maximize accessibility to the target embedded in chromatin. A cell cycle study showed that the highest level of targeted crosslink formation was in S phase cells. Cellular processing of the targeted psoralen crosslink can be error free or may have mutagenic consequences, specifically deletions or base substitutions. We have determined the frequency of these endpoints in wild type and DNA repair deficient cells. Our results support a model of crosslink repair in which multiple branching pathways are available for crosslink metabolism. The pathway that leads to base substitution mutagenesis is independent of the pathway that results in deletion mutagenesis. Our targeting strategy also has practical implications. We have recently shown that the pso-TFOs can direct knock in of an exogenous DNA sequence by targeted homologous recombination at frequencies that are 1000-10,000 greater than in the absence of the pso-TFO. We are currently exploring the potential of this strategy for "in situ" correction of mutations in cells from individuals with genetic disorders. Recently we have developed another strategy, based on immunofluorescence and confocal microscopy, for following crosslink repair. We have synthesized psoralens with immunogenic tags. Incubation of cells with these compounds followed by photoactivation introduces tagged crosslinks into the genome. The crosslinks can be localized by laser induced photoactivation of a defined region in the nucleus of living cells. We have used this approach to follow crosslink persistence in repair proficient and deficient cells, and also to track the recruitment of repair proteins to the crosslinks. |
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| Section on Gene Targeting (left to right). Row 1: Jilan Liu, Hong Li; row 2: Jia Liu, Parameswary A. Muniandy, Rebecca Greaves; row 3: Alokes Majumdar, Michael Seidman. Not shown: Andrey Semenyuk. |
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