Structural insight into the exonuclease activity of Apurinic/Apyrimidinic Endonuclease 1 in DNA processing
| Autor: | Liu, Tung-Chang, 劉東璋 |
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| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 Ape1 (Apurinic/apyrimidinic endonuclease 1) is a multifunctional protein. Dysfunctional Ape1 leads neurodegenerative diseases or cancer. Ape1 is composed of N-terminal redox domain and C-terminal nuclease domain. The N terminal redox domain with redox activity is involved in the regulation of cell proliferation, cell growth, or apoptosis. The C terminal nuclease domain with endonuclease activity is responsible for AP site (Apurinic/apyrimidinic site, a nucleotide without nitrogen base) nicking in base excision repair. Interestingly, the nuclease domain is also a homolog of E.coli ExoIII, which possesses strong 3’ – 5’ exonuclease activity. Thus, Ape1 has been reported as an exonuclease and is likely participated in DNA proofreading or apoptosis. However, the molecular mechanisms and related cellular functions of Ape1 exonuclease activity remain unclear. In order to solve above questions, we determined two crystal structures which bind dsDNA from terminus, including Ape1-dsDNA-1nt 5’-overhang complex and Ape1-dsDNA-2nt 5’-overhang complex. Our crystal structures and biochemical assays shown mApe1 contains exonuclease activity and prefers cleaving dsDNA. Loss of exonuclease activity in mutant mApe1 (E95A or H308A) depicts that catalytic center of endonuclease activity and exonuclease activity in Ape1 are similar. In addition, we also identified the potential working partner of Ape1. Ape1 and TREX1 (Three-prime repair exonuclease 1) both are members of SET complex on ER membrane. By using pull-down and nuclease activity assays, we demonstrated that Ape1 interacts with TREX1 directly and works cooperatively with TREX1 in dsDNA processing. In conclusion, we not only provide the structural insight into the exonuclease activity of Ape1 but also identify the working partners of Ape1 to clarify the roles of Ape1 in DNA processing. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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