Mitotic stability of genomes: From initiation to resolution of homologous recombination – IniToResGenStab

Maintenance of genome integrity is crucial for cell survival during somatic growth. Homologous recombination is an important player in this process. It is essential for programmed double-strand break repair that is triggered in meiosis or during yeast mating-type interconversion. Homologous recombination is also involved in the processing of strand breaks (single or double) that are formed accidentally during the cell cycle. However, unrestrained homologous recombination can also lead to the formation of toxic intermediates or potentially harmful genome rearrangements, the hallmark of certain types of cancer. The mechanisms of homologous recombination are ubiquitous and have been studied extensively in many different species ranging from bacteria to man, including archaea and yeast. The Mre11/Rad50 complex is well conserved through evolution and plays a key role in double-strand break repair by homologous recombination and end-joining. Despite this central role, the 'modus operandi' of this complex is still not fully understood. In vitro, this complex exhibits a tethering function that maintains DNA ends close to the nuclease activities. However, these activities do not explain the end trimming functions carried out in vivo by this complex that promote recombinase loading onto single-stranded DNA. This suggests that it could associate with other partners to perform this task. The later steps of the recombination process (following homology search and strand-invasion) are not well-characterized but involve the activities of the Sgs1 and Srs2 helicases. Even less is known about the resolution enzymes in eukaryotic cells. The aims of this project are to define and characterize players in the early and late steps of homologous recombination: On one hand, the initiation step will be studied in hyperthermophilic archaebacteria. In these organisms, two putative partners of the Rad50/Mre11 complex have been identified recently in the laboratory of C. Elie. These partners form a novel family of proteins. NurA is a nuclease that degrades DNA from 5'→3', the direction opposite to that of the exonuclease activity associated with Mre11, whereas HerA is a bipolar DNA helicase that is capable of unwinding DNA from a 3' or 5' overhang. Recent results suggest that Mre11/Rad50 and NurA/HerA could play an important role in the initiation of homologous recombination and could explain how protruding 3'OH single-strands are produced. We now want 1) to determine the molecular mechanism, the structure and the function of NurA and HerA 2) to get new insights on the molecular mechanism and thus, in the function of the Rad50-Mre11 complex and 3) to establish the functional interactions between these proteins and eventual other partners. Furthermore, the resolution of the NurA and HerA structures might help in the search for functional analogues in Eucarya. On the other hand, to understand the later steps of homologous recombination, we propose to tackle several aspects in the yeast Saccharomyces cerevisiae. We will 1) identify and characterize the toxic intermediates formed during normal metabolism and revealed in the absence of Srs2, 2) identify new mutants of Srs2 and characterize them genetically and biochemically, 3) use a Polδ mutant affected only in repair synthesis to understand the involvement of DNA polymerases in recombinational repair and 4) identify activities involved in Holliday junctions resolution. This research project relies on complementary approaches of classical and molecular genetics, biochemistry and electron microscopy. To achieve this goal, three laboratories are involved: partner 1 is specialized in the genetics and biochemistry of homologous recombination; partner 2 is specialized in the recombinational repair in hyperthermophilic archaea and partner 3 is specialized in the electron microscopy analyses of nucleoprotein complexes involved in recombination and ties both parts of the project together.