DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. In eukaryotes, two proteins, Mre11 and Rad50 play a major role in DSBs recombination/repair. In particular, these proteins are involved in the maturation of DSBs during homologous recombin ation which requires 3' overhangs for recombinases. However, their in vitro activities cannot explain this maturation mechanism suggesting the existence of additional partners. In thermophilic archaea homologs of Mre11 and Rad50 are organised in an operon-like structure, together with two genes encoding a nuclease and a helicase. The nuclease (NurA) exhibits 5´-3´ exonuclease activity, indicating that it could be essential for the production of the 3´overhangs (Constantinesco et al., 2002). I´m interested i n the design of an in vitro system for DSBs maturation by the Rad50-Mre11 pathway. In the proposed study I will analyse the interactions between the four proteins Mre11, Rad50, NurA and HerA and investigate the activities of the proteins as well as of the potential complexes. Subsequently I will identify additional proteins binding to the complexes using crude protein extracts from Sulfolobus and specific antibodies. In addition, I will initiate the study of in vivo functions using recently developed geneti c tools for Sulfolobus. Together this should lead to a comprehensive model of the initiation step of DNA recombination/repair in archaea.
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