University of Virginia Health System

Biochemistry, Molecular Biology, and Genetics
Cell and Developmental Biology
Our laboratory is interested in the control of mammalian chromosomal replication. It has been known for a long time that DNA replication initiates at multiple sites termed "origins of replication" along each DNA fiber. However, the precise location or properties of even a single origin had not been determined. Several years ago, we localized the first mammalian origin, which lies within the 55 kb spacer region between the DHFR and 2BE2121 genes. This spacer is characterized by the presence of a centered matrix attachment region (MAR). Using a two-dimensional gel electrophoretic technique to precisely identify initiation sites, we have shown that the origin corresponds to a very broad zone of potential sites scattered throughout the intergenic region. There are several genetic elements whose functions could contribute to the regulation of origin activity in this locus (e.g., a replicator, the promoters of the two flanking genes, the MAR). To identify the responsible controlling elements, we have devised a novel homologous recombination strategy to specifically knock out or mutagenize any fragment within the intergenic region or the two flanking genes. Our recent studies suggest that both the promoter and the 3' end of the DHFR gene are required for proper origin function. Surprisingly, however, the MAR is required to effect sister chromatid separation shortly after the origin fires. Our long-range goals are to define the initiation reaction in molecular terms by identifying both the cis- and trans-regulatory elements that participate, and to reconstitute initiation in vitro.

To gain insight into whether the DHFR origin is typical of other mammalian origins, we have recently devised a very efficacious strategy for isolating all of the active origins in the human genome. The resulting library has been shown to be essentially pure. By comparing the sequences of the origin clones to the human genome database, we will be able to determine their distribrutions vis-a-vis active genes, and whether or not they share common sequence motifs.
Cancer Research - Molecular Medicine
We are interested in the mechanism by which cells amplify DNA. DNA sequence amplification is an important phenomenon that only occurs in tumor cells, which usually lack critical damage-sensing pathways. Most human tumors have amplified one or more cellular oncogenes, which are thought to confer a selective growth advantage over surrounding normal cells. Thus, it is important to determine how gene amplification occurs. In fluorescence in situ hybridization studies, we have shown that the very first amplification events are mediated by chromosome breaks, followed by sister chromatid fusion, bridge formation, and further breaks. Thus, it is now clear why cells that lack the ability to sense DNA damage (i.e., breaks) are able to amplify oncogenes, while normal cells cannot. We are devising new strategies to examine the products of the earliest amplification events at the nucleotide sequence level to determine why and how cell breaks and fusions occur. Our goal is to identify steps in the amplification process that could be targets for chemotherapy.

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http://www.people.virginia.edu/~jlh2d