Functions and Dynamics of DNA Repair Proteins in Mitosis and Meiosis
My PhD project encompassed studies on the functions of several different proteins, all involved in DNA repair, in somatic and germ-line cells. Hr6b and Rad18Sc are involved in a DNA repair mechanism called â€˜Replicative Damage Bypassâ€™ (RDB), and function as ubiquitin conjugating enzyme and ubiquitin ligase, respectively. In Hr6b knockout mice, spermatogenesis is severely impaired. Analysis of Hr6b knockout testis has indicated that Hr6b plays a role in meiotic recombination and in post-meiotic chromatin remodelling. Both these functions appear not to be directly related to RDB. We partially rescued the Hr6b knockout phenotype using a transgenic approach to express Hr6b-GFP and Hr6b-HA. Furthermore, we found that Rad18Sc, colocalizes with Hr6b on unpaired and transcriptionally inactive chromosomal regions, such as the X and Y chromosome in pachytene spermatocytes. These data indicate that Rad18Sc may have a function in meiotic prophase, outside the contex! t of RDB. Rad51 is a key player in homologous recombination, which is one of the major pathways for repair of DNA double-strand breaks (DSBs). To study Rad51 function in living mitotic and meiotic cells, we targeted mouse ES cells with a DNA construct in which endogenous Rad51 is replaced by LoxP flanked Rad51-GFP. However, Rad51+/GFP ES cells proved to be more sensitive to DNA damaging agents than wild-type ES cells. Still, in Rad51+/GFP cells, Rad51-GFP is nuclear and forms spontaneous foci during S phase and following g radiation, indicating that this fusion protein shows similar subcellular localization properties compared to endogenous Rad51. Currently, we are studying the intracellular dynamics of Rad51-GFP by fluorescence correlation spectroscopy (FCS) measurements and fluorescence recovery-after-photobleaching (FRAP) experiments. In another part of my project, we studied the role of another DSB repair protein named Rad54 in spermatogenesis. Two mouse (and human) paralogs of yeast RAD54 have been identified: Rad54 and Rad54B. Both proteins show relatively high expression in testis. We developed a new technique for live cell and tissue imaging in testis tubules and used it to study the localization and dynamics of Rad54-GFP in different germ cell types of the testis. Rad54-GFP was detected at high levels in proliferating spermatogonia, but absent from leptoten/zygotene spermatocytes. Rad54-GFP signal reappeared briefly in mid-late pachytene spermatocytes. During homologous recombination (HR) in somatic cells, Rad54 is involved in the pre-synaptic, synaptic and post-synaptic phases of HR. To evaluate possible involvement of Rad54 and Rad54B in meiosis, we studied localization of Rad51 in spermatocytes from Rad54 and Rad54B knockout and double-knockout mice. In these single and double-knockout spermatoc! ytes, we found normal accumulation of Rad51 foci during early meiotic prophase (leptotene/zygotene), but abnormal Rad51 localization in spatially restricted large clusters was found in pachytene and diplotene spermatocytes, when Rad51 foci normally disappear in the wild type. These studies suggests that Rad54 is probably not involved in the pre-synaptic and synaptic steps of meiotic HR, but is required for disassembling Rad51 during the post-synaptic phase.
|Keywords||DNA repair, Hr6b, Rad18, Rad51, Rad54, fluorescence, meiosis, testis|
Uringa, E.J.. (2005, September 14). Functions and Dynamics of DNA Repair Proteins in Mitosis and Meiosis. Retrieved from http://hdl.handle.net/1765/6935