Double-strand breaks (DSBs) are highly cytotoxic DNA lesions that are usually repaired by two major mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). HR is initiated by 5'-3' resection, generating 3' single stranded DNA tails coated by Replication protein A (RPA), which can be used in later steps for homology search and repair. The 5'-3' resection step is a critical determinant of repair pathway choice that commits cells to HR instead of NHEJ, and it's also required for DNA damage checkpoint activation. Studies in the budding yeast Saccharomyces cerevisiae have shown that the conserved Mre11-Rad50-Xrs2 (MRX) complex, together with Sae2, initiates end resection while more extensive processing of 5' strands requires the 5'-3' exonuclease Exo1, or the combined activities of the Sgs1 helicase and Dna2 endonuclease. In this thesis we will discuss the function of RPA and Sae2 based on our experimental observations. RPA is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism.

It has been shown in vitro that RPA directly participates in end resection by stimulating the Sgs1 helicase and Dna2 endonuclease. To investigate the role of RPA for end resection in vivo, we used a heat-inducible degron allele (td-RFA1) that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. Complete loss of RPA resulted in a defect in both the Exo1 and Sgs1-Dna2 extensive resection mechanisms, while resection initiation by MRX-Sae2 was unaffected. Interestingly, Dna2 was unable to localize to DSBs in the absence of RPA, whereas Exo1 localization was unaffected indicating that the role of RPA in the resection pathways is distinct. The short single-stranded DNA tails formed in the absence of RPA were unstable, represented by 3' strand loss and formation of foldback hairpin structures. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements. While Mre11 possesses 3'-5' dsDNA exonuclease and ssDNA endonuclease activities, Sae2 was reported to activate its endonuclease activity, which initiates end resection.

We identified mre11-P110L and four more mutants from a screen that bypass Sae2 for camptothecin (CPT) and MMS resistance. None of them restored endonuclease activity, neither did they improve resection. Persistent Mre11 foci and hyper-checkpoint signaling caused by sae2Δ upon DNA damage was suppressed by mre11-P110L. These findings demonstrate that the DNA damage sensitivity of sae2Δ is not caused by defective resection, but by failure to remove MRX from ends and switch off checkpoint.