MOLECULAR STUDIES OF HUMAN GENETIC RECOMBINATION

基因信息的重新排列

The rearrangement of genetic information through the recombination of DNA occurs in all forms of life. Genetic recombination allows gene mapping and has been implicated in immunoglobulin gene rearrangement, DNA repair, gene amplification, the initial development of cancer, and the prevention of aneuploidy with its resultant birth defects. Despite the importance of recombination in higher eukaryotes, it has been difficult to study and little is known about its biochemistry. In this proposal a central step in recombination, strand transfer between homologous DNAs, will be studied. I previously reported the first characterization of a mammalian strand transfer protein (recombinase). I now propose a detained study of the strand transfer reaction promoted by this protein. Analyses will focus on a) the formation of nucleoprotein networks by the recombinase and the nature of its search for homology, b) the formation of paranemic joints and c) the directionality of joint formation and branch migration. The effect of special DNA sequences on the strand-transfer reaction will also be determined by the use of novel substrates and oligonucleotides. In parallel with the above studies, a new method for measuring recombination in human cells in culture with be developed. This system utilizes tandem, complementary mutant antibiotic- resistance genes contained in retroviral vectors. It will be used to measure extrachromosomal recombination, intrachromosomal recombination, interchromosomal recombination (sister chromatid exchange) and gene conversion in normal fibroblast lines and fibroblast lines derived from patients with chromosome instability syndromes. Recombination mutants may be detected by these experiments. The identification of such mutants would aid the analysis of recombination in mammalian cells and may also provide a unique opportunity to study the molecular pathology of these diseases.