DNA CRUCIFORMS AND HUMAN DISEASE

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As a Procter Scholar, Dr. Bissler identified mutations in the C1 inhibitor (C1INH) gene from 40 kindred with hereditary angioneurotic edema. He found two mutation hotspots which appear to share intrinsic DNA replication infidelity. The mutation hotspot he will investigate as part of his studies outlined here is in exon 8 of the C1INH gene. This region forms a thermodynamically stable stem loop structure. Similar structures are found in the Antithrombin III (ATIII) human immunodeficiency virus TAR and platelet drive growth factor A genes. Both ATIII and C1INH also cluster point mutations in the sequence which engages in these cruciform structues. He will study the ability of these genes to form cruciform structures and examine the effect of these cruciforms on eukaryotic replication machinery. Currently, the methods for studying intrinsic mutation mechanisms are labor intensive requiring extensive sequence analysis. Dr. Bissler has designed a system which has the potential to selectively isolate only bacteria harboring mutant plasmid which has undergone frameshift or point mutagenesis. This method will greatly reduce the labor required to access the putative mutagenicity of the cruciform from various human disease associated genes. By combining the information from the mutation frequencies and eukaryotic replication machinery pausing, Dr. Bissler will more clearly define the role of replication pausing in mutagenesis. Better understanding of the factors involved in intrinsic DNA instability is important to elucidate the basic mechanisms of mutagenesis. The observations from the proposed research will bear directly on the creation of stable constructs which might be used for gene therapy. Dr. Bissler's current environment is uniquely suited to support his continued career development. He has been provided with his own office and laboratory space. The laboratory contains the equipment needed for his studies, and he has access to core equipment at the Children's Hospital Research Foundation, includes state of the art oligonucleotide synthesis facilities. Dr. Bissler will be financially supported by the Research Foundation to travel to Houston in order to learn techniques from Dr. Sinden, a consultant. Dr. Bissler's snsor, Dr. Kathieen Dixon, has had extensive experience in assisting young investigators develop independent careers in laboratory investigation and has scientific expertise in areas that Dr. Bissler proposes to study. Dr. Bissler also will have ample opportunity to further his basic science knowledge by participating in journal clubs, seminars and classes.