Structural and Biochemical Investigation of the Molecular Mechanisms of Human Exonuclease I and Characterization of Its Protein-Protein Interactions

Human exonuclease 1 (hExo1) is a RAD2/XPG family 5’ structure-specific nuclease and is important for various central DNA metabolic processes including replication, recombination, and repair. Its defects disrupt genome integrity and are associated with over ten different human cancers. hExo1 can process a variety of DNA/RNA substrates including blunt-end, recessed-end, nicked, gapped, and 5' flaps. Its dominant, processive 5'-3' exonuclease activity creates long single strand DNA. The secondary 5' flap endonuclease activity specifically removes 5’ flaps. Both reactions are catalyzed by the active site that is highly conserved in the RAD2/XPG family. To understand this multi-function protein, it is crucial to decipher the molecular mechanism of its catalysis. Although the crystal structures of the hExo1 catalytic domain were published, the reaction mechanism of this enzyme and other RAD2/XPG enzymes was still under debate, even the active site geometry during catalysis was unclear.

Moreover, even less was known about the hExo1 processing of 5’ flaps. It was unclear how hExo1 recognizes the endonucleolytic scissile bond and accommodates the bulky 5’ single strand DNA to the active site. Besides, hExo1 can also cleave 5’ flaps exonucleolytically, but the intermediate steps that lead to the two distinct cleavage patterns were still unknown. And thus, the molecular mechanism of 5’ flap binding and cleavage remained one of the most controversial questions of the RAD2/XPG family.

While the hExo1 catalytic domain is essential for its activity, hExo1 C-terminal region modulates its activity by interacting with different protein partners and recruits hEox1 to different DNA repair pathways. The interaction interfaces are separated by several hundred residues from the hExo1 active site, which raise questions about the mechanism of activity modulation.

A combined biochemical and structural approach has been deployed to study these long-lasting questions. By developing a system to initiate enzyme reactions in crystallo, hExo1 reaction intermediates have been trapped that reveal structures of various steps of both exo- and endonucleolytic cleavage with multiple substrates. These structures provide detailed illustrations of the five-stage process of hExo1 processive excision, and the steps of both exo- and endonucleolytic cleavages of 5’ flaps. And thus, these results reveal the consecutive, interlocking conformational changes of the protein-DNA complex, which guide different substrate into the active site and cleaved by the same two-metal catalytic mechanism. The observed global motions and local rearrangements also provide the foundation for the processivity, fidelity, and regulation of hExo1 reaction. A stochastic self-regulatory mechanism was also discovered in this study, which modulates hExo1 reaction at different stages of the reaction cycle. Moreover, a series of molecular cloning, biochemical assays and structural methods have been employed to study hExo1 interactions with its protein partners. Novel interactions have been discovered and characterized, which leads to the development of new hypotheses about hExo1 activity modulation and pathway regulation.

Taken together, results presented in this dissertation offer comprehensive solutions to the long-lasting debates about the reaction mechanisms of the RAD2/XPG family proteins. This work also provides new insights about hExo1 activity modulation, which extends the knowledge about the RAD2/XPG family nucleases. These results also further advance the understanding of hExo1 interaction network and provide new insights into the function and regulation of hExo1 in different pathways, which expands the knowledge about multiple DNA metabolic pathways and related diseases.