Meiotic and mitotic functions of mammalian RAD 18 in DNA double-strand break repair

This thesis focuses on the role of RAD 18 in DNA double-strand break (DSB ) repair. Much is known about the role of RAD 18, and its critical substrate PCNA in replication damage bypass (RDB ) repair. However, the roles of RAD 18 in DSB repair are still elusive, although several interaction partners of RAD 18 have been identified, and the radiation-sensitivity of Rad18 knockout cells has shown that this E3 ligase is active in DSB repair. First, a general introduction on the possible involvement of RAD 18 in DSB repair mechanisms and RDB that operate in mitotic and meiotic cells is presented in Chapter 1. In Chapter 2, we examined the dynamic localization of human RAD 18 during the cell cycle and after DNA damage in living cells. The DNA damage response functions of RAD 18 after different types of damages (UV and IR) were analyzed. In order to distinguish DSB repair functions of RAD 18 from functions in the RDB pathway, the dynamics of the known substrate of RAD 18 in RDB , PCNA , was also examined in living cells. Subsequently, we performed a structure-function analysis of RAD 18 and examined the requirements for RAD 18 localization to DSB s in mitotic cells. We also show that RAD 18 facilitates recruitment of the checkpoint protein RAD 9 to DSB repair sites (Chapter 3). Next, we examined the role of mouse RAD 18 in meiotic DSB repair by analysing the testicular phenotype of Rad18 knockdown mice (Chapter 4). Also, the function of PCNA modification in meiosis was examined in Chapter 5. We propose a possible role of SU MOylated PCNA in meiotic crossover control. Based upon our observations on RAD 18 localization to DSB s in meiosis, we have performed a detailed analysis of the accumulation of DSB -repair proteins in the presence and absence of meiotic (SPO 11-induced) DSB s in spermatocytes, in relation to chromosome pairing (Chapter 6). Finally, we present a general discussion of the results described in Chapter 2 - 6 and present a model of RAD 18 functions in DSB repair (Chapter 7).

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