First steps in homologous recombination: DNA end resection
Research area: Recombination Mechanisms
Group leaders: Petr Cejka
- Elda Cannavò Cejka, Scientist
- Sean Michael Howard, Scientist
- Maryna Levikova, University of Zurich
- Cosimo Pinto, University of Zurich
Status: In progress
Homologous recombination is initiated by the nucleolytic degradation (resection) of the 5'-terminated DNA strand of the DNA break. This leads to the formation of 3'-tailed DNA, which becomes a substrate for the strand exchange protein RAD51 and primes DNA synthesis during the downstream events in the recombination pathway. DNA end resection thus represents a key process that commits the repair of DNA breaks into recombination. Research from multiple laboratories established that DNA end resection is in most cases a two-step process. It is initiated by the nucleolytic degradation of DNA that is at first limited to the vicinity of the broken DNA end. This is carried out by the Mre11-Rad50-Xrs2 (MRX) complex and Sae2 proteins in yeast, and MRE11-RAD50-NBS1 (MRN) and CtIP proteins in human cells. We could reconstitute these reactions in vitro, and demonstrated that Sae2 and CtIP stimulate a cryptic endonuclease activity within the yeast MRX or human MRN complex, respectively. The activity of Sae2/CtIP is absolutely dependent on its phosphorylation. The reconstituted DNA clipping reaction allows us to investigate the mechanism of this process as well as its regulation by posttranslational modifications and additional protein co-factors.
Downstream of MRX-Sae2 and MRN-CtIP, which process only a limited length of DNA, DNA end resection is further catalyzed by Sgs1-Dna2 or Exo1 in yeast and BLM-DNA2, WRN-DNA2 or EXO1 in human cells. We are interested how the functions of these factors integrate in protein complexes to form molecular machines that are uniquely capable to resect long lengths of DNA, which is required for homologous recombination. We are specifically interested in the Dna2 enzyme, and could show that both yeast Dna2 and human DNA2 possess a cryptic helicase activity. We now investigate how the motor activity of Dna2 promotes DNA end resection, as well as how it is regulated in cells. Finally, as some of these enzymes are upregulated in various human cancers, we are also searching for small molecules capable to inhibit these pathways.