The structure and mechanism of the EXD2 nuclease in DNA repair

Abstract

DNA double strand breaks (DSBs) are one of the most toxic DNA lesions. They can jeopardise stability of the genome and lead to cancer development. There are two main pathways of DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). EXD2 is a recently identified 3'-5' exonuclease, which promotes HR by facilitating DNA end resection. It has been established that the MRN complex (MRE11, RAD50, NSB1), as well as EXD2, are required for efficient HR. However, the exact contribution of EXD2 in HR and other DNA pathways, is unknown. To elucidate the molecular mechanism of DNA end resection and the role of EXD2 in this process, I optimized purification of full-length hEXD2 expressed in insect cells and performed biochemical characterization of EXD2 activity in vitro. EXD2 exhibits 3'-5' exonuclease activity on multiple DNA structures, including 3' phosphorylated DNA, mismatched nucleotides, hairpins, tolomeric G-quadruplexes, gapped DNA and structures mimicking active and reversed replication forks. This data suggests that EXD2 may function to 'clean' modified DSBs to enable their efficient repair. It can also process non-canonical DNA structures, facilitating DNA replication. Moreover, RPA inhibits EXD2 activity on ssDNA ends and decreases digestion efficiency on substrate with an accessible dsDNA end. Interestingly, this thesis reports the first observation of EXD2's weak endonuclease activity. Generation of a novel (H442A, H463A) mutant highlights the importance of HNH domain, which is present in many endonucleases. A preliminary 3D structure of full-length EXD2, obtained by Fabienne Beuron, shows that both exonuclease and NHN-domains are located close to a tunnel, which may be an entry site for DNA substrates.