In this application, Dr. Engels proposes to continue his studies on the biology of P-elements and the mechanisms of gap repair. Seven different lines of investigation are described. The first is a test of the synthesis dependent strand annealing (SDSA) model for DNA gap repair. In this model, both ends of the gap invade their templates independently to synthesize complementary strands. In those circumstances in which there are two potential repair templates, both templates could in principle be used for strand synthesis. If the primary mechanism for gap repair follows this SDSA pathway, then it should be possible to observe bi-template repair events when there are two potential templates present. Moreover, this bi-template repair should occur at a frequency consistent with the SDSA model. In the second line of investigation, Dr. Engels proposes experiments aimed at understanding the parameters governing the search for homologous sequences. In previous studies he found that there is a six- fold increase in the conversion frequency when the potential template sequence was on the same chromosome. Experiments will include determining whether this cis effect is also observed when the potential templates are on different arms of an attached-X chromosome, or on other rearranged chromosomes containing, for example, intervening heterochromatic regions and whether the frequency of conversion depends upon the position of two potential template relative to the gap, i.e., is the template closer to the gap preferentially used. In the third set of experiments, Dr. Engels will attempt to identify genes that may participate in the gap repair process. The first locus to be examined is spellcheck (spel1), a Drosophila homolog for the yeast and human MSH2 mismatch repair genes. Dr. Engels will determine if this gene is essential for viability, and whether mutations in it have any effect on the gap repair process. He also plans to ectopically express a MSH2 homolog from another species. Studies in other organisms indicate that ectopic expression of a foreign MSH2 can result in a dominant mutator phenotype. In other experiments Dr. Engels will determine whether mutations known to cause defects in DNA repair also show alterations in double-strand break repair. These genes include mei-41, mei-9, top1, and mus309. In addition to measuring the frequency of conversion events, a number of other parameters (e.g., the length of the conversion tracts, the use of ectopic templates) will be examined. In the fourth set of experiments, Dr. Engels will examine gap repair of DNA breaks generated by the yeast HO system. Transgenes carrying a heat inducible HO endonuclease gene and the target sequence for the endonuclease will be introduced into flies. The repair of HO-induced breaks will then be compared with those induced by P-elements. The fifth section of the proposal describes continuing efforts to optimize the use of P-induced gap repair as a method for gene replacement.
The aim of the experiments described in section six is to examine the site specificity of P-element insertion, in particular what defines an insertional hot spot. Single base changes will be introduced into as well as in the immediate vicinity of an insertional hot spot in the white gene. Dr. Engels will then determine what effects these nucleotide changes have on the frequency of (somatic or germline) P- element insertion. The experiments described in the last section of the proposal are aimed at understanding the relationship between P-element transposition and increased recombination. Dr. Engels suggests that the recombination is a consequence of the gap repair process and is initiated by unligated nicks at the edges of the conversion tracts. A P-element insertion at 50C will be used to examine the recombination frequency (and conversion tract distribution) as a function of distance from the insertion site.
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