Publications

To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the FEN-1 gene. The mutants were then expressed in Escherichia coli and purified using a His-tag. Even though the mutants bind to the flap DNA to different degrees, most of the mutants lost flap nuclease activity with the exception of an E160D mutant. This mutant retained wild type-like binding ability, specificity, and partial catalytic activity. Detailed steady state and pre-steady state kinetic analysis revealed that the functional deficiency of this mutant was due to retardation of the endonucleolytic cleavage. When the mutant enzyme E160D was expressed in yeast, it partially complements the biological functions of the homologous yeast gene, RAD27, and reverses the hyper-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity.

The matrix metalloproteinase gelatinase A plays a central role in several critical physiologic processes, including angiogenesis, tumor invasion/metastasis, and chronic inflammation. We demonstrate that high level gelatinase A expression is mediated by a unique interaction of two developmentally regulated transcription factors, AP2 and YB-1, within a discrete 40-base pair enhancer element (RE-1) located in the 5'-flanking region of the gelatinase A gene. Electrophoretic mobility shift assay studies and immunoprecipitation experiments confirmed a direct interaction of AP2 with this binding sequence in the form of AP2.YB-1 heteromeric complexes. Binding of AP2.YB-1 complexes to the RE-1 sequence results in the formation of extended single-stranded DNA regions and may stabilize DNA conformational changes. Overexpression of YB-1 and AP2 proteins by gelatinase A synthesizing hepatoma HepG2 cells induced a synergistic increase in the RE-1-mediated transcription of nearly 160-fold. Thus, the transcription of gelatinase A is subject to a previously unrecognized interplay of double (AP2) and single-stranded (YB-1) DNA binding transcription factors to yield a highly regulated pattern of gene expression.

Introduction of the MHC class I transgene H-2Dd on C57BL/6 (B6) background conveys NK cell-mediated "missing self" reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2Dd/Ld MHC class I transgenes, and show that the alpha1/alpha2 domains of H-2Dd were necessary and sufficient to induce "missing self" recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the alpha1/alpha2 domains of H-2Ld induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the alpha1/alpha2 domains of H-2Ld on NK cell specificity was surprising, considering that both H-2Ld and H-2Dd have been reported to interact with Ly49G2. Therefore, the role of H-2Ld for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2Dd, but not H-2Ld, abolished killing by sorted Ly49G2+ NK cells, indicating that H-2Ld does not inhibit NK cells via the Ly49G2 receptor.