Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Bruce D. McKee
Ranjan Ganguly, Kwang W. Jeon, Mary Ann Handel, John Koontz
Among proteins involved in homologous recombination, Rad51 is an essential enzyme possessing DNA binding, ATPase, and strand transfer activities. The fu nctions of Rad51 in repair of double-strand breaks (DSB) and meiotic recombination have been extensively studied in yeast and mammals, but little is known about its roles in cell cycle regulation and apoptosis. In this study, we examine the roles of Rad51dm protein in Drosophila melanogaster.
Like other Rad51 proteins, Rad51dm is involved in DNA repair and recombination. The function of Rad51dm in DNA repair was demonstrated by examining the sensitivity of transgenic animals to methyl methanesulfonate (MMS) that induces double-strand breaks in DNA. When treated with MMS , transgenic larvae carrying extra copies of the Rad51dm gene showed higher survival rates. To down-regulate the function of native Rad51dm, hsp26-IRrad51 transgenic animals carrying Rad51dm gene in an inverted-repeat orientation ( IR) driven by a heat shock inducible promoter, hsp26, were generated and the MMS sensitivity of these lines were determined . Under a heat shock condition, the survival rates of hsp26-IRrad51 larvae were greatly reduced compared to that of the yw control group. In addition, the transcript level of Rad51dm in wild-type flies increased 2- to 3-fold after X-ray or MMS treatment. These results suggest that Rad51dm is involved in repair of DNA damage.
To study if Rad51dm is involved in meiotic recombination, we determined non-disjunction rates of sex chromosomes and frequencies of recombination using hsp26-IRrad51 females. Under heat shock, hsp26-IRrad51 females showed elevated frequency of X-X non-disjunction and reduced meiotic recombination events compared to control groups without heat shock, implying that IR Rad51dm RNA may inhibit the function of Rad51dm in meiotic recombination. However, non-disjunction rates of X-Y chromosomes of hsp26- IRrad51 transgenic males were similar between the heat-shock group and non-heat-shock group, suggesting that Rad51dm may not be involved in male meiosis.
To determine the expression level and the distribution of Rad51dm protein, we generated a polyclonal antibody. lmmunoblot analysis revealed that Rad51dm protein was highly expressed in embryos, but hardly detectable in any other tissues. lmmunostaining of embryos showed that the Rad51dm foci associated with unknown materials stained with 4'6-diamidino-2-phenylindole (DAPI), but they did not interact with mitotic chromosomes. To investigate a role of Rad51dm in meiosis, we examined spermatocytes. Although Rad51dm foci were not detected in wild-type spermatocytes, overexpressed Rad51dm proteins exclusively localized in the nucleus during prophase I and rapidly disappeared throughout the subsequent stages as observed for mammalian RAD51 proteins. Since the meiotic recombination occurs only in female of Drosophila melanogaster, it will be interesting to study the distribution of Rad51dm protein in oocytes.
To investigate the overexpression effect of Rad51dm protein, a heat-shock inducible promoter and the UAS-GAL4 binary expression system were used to generate hsp26p-rad51 and UAS-rad51 transgenic flies. Both transgenic lines showed that the overexpression of Rad51dm protein results in lethality regardless of heat shock treatment. Immunocytochemistry revealed that Rad51dm foci in wing and eye imaginal discs are colocalized with foci stained by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, suggesting that the overexpression of Rad51dm protein induces apoptosis. In addition, crosses of UAS-rad5 1 and UAS-reaper with various GAL4 lines showed that the phenotypes caused by overexpression of Rad 51dm protein are similar to those of Reaper protein, a proapoptotic protein, suggesting that Rad51dm might function in apoptosis. To determine if Rad51dm affects cell cycle progression , eye imaginal discs were stained with the mitotic-specific anti-phospho-histone H3 antibody. The result revealed that overexpression of Rad51dm protein disrupts the normal mitotic pattern in the second mitotic wave in eye discs. In conclusion, it is likely that Rad51 may act as a 'sensor' molecule that monitors DNA damage and determine cell survival or cell death.
Yoo, Siuk, "Functional Analysis of Drosophila Rad51 Protein in DNA Repair, Recombination, and Apoptosis. " PhD diss., University of Tennessee, 2001.