Browsing by Author "Chu, Lee-Ya"

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Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN-4 Bound with 2-Morpholin-4-ylethanesulfonate (MES)
(2016-09-08)
Huang, Kuan-Wei
;
Hsu, Kai-Cheng
;
Chu, Lee-Ya
;
Yang, Jinn-Moon
;
Yuan, Hanna S.
The DEDDh family of exonucleases plays essential roles in DNA and RNA metabolism in all kingdoms of life. Several viral and human, DEDDh exonucleases can serve as antiviral drug targets due to their critical roles virus replication. Here using RNase T and CRN-4 as the model systems, we identify potential inhibitors for DEDDh exonucleases. We further show that two of the inhibitors, ATA and PV6R, indeed inhibit the exonuclease activity of the viral protein NP exonuclease of Lassa fever virus in vitro. Moreover, we determine the crystal structure of CRN-4 in complex with MES that reveals a unique inhibition Mechanism, by inducing the general base His179 to shift out of the active site. Our results not only provide the structural basis for the inhibition mechanism but also suggest potential lead inhibitors for the DEDDh exonucleases that may pave the way for designing nuclease inhibitors for biochemical and biomedical applications.
Identification of inhibitors for the DEDDh family of exonucleases and a unique inhibition mechanism revealed by crystal structure analysis of CRN-4 bound with MES
(2016-01-01)
Hsiao, Yu-Yuan
;
Huang, Kuan-Wei
;
Hsu, Kai-Cheng
;
Chu, Lee-Ya
;
Yang, Jinn-Moon
Structural basis for overhang excision and terminal unwinding of DNA duplexes by TREX1
(2018-05-01)
Huang, Kuan-Wei
;
Liu, Tung-Chang
;
Liang, Ruei-Yue
;
Chu, Lee-Ya
;
Cheng, Hiu-Lo
Three prime repair exonuciease 1 (TREX1) is an essential exonuclease in mammalian cells, and numerous in vivo and in vitro data evidenced its participation in immunity regulation and in genotoxicity remediation. In these very complicated cellular functions, the molecular mechanisms by which duplex DNA substrates are processed are mostly elusive because of the lack of structure information. Here, we report multiple crystal structures of TREX1 complexed with various substrates to provide the structure basis for overhang excision and terminal unwinding of DNA duplexes. The substrates were designed to mimic the intermediate structural DNAs involved in various repair pathways. The results showed that the Leu24Pro25-Ser26 cluster of TREX1 served to cap the nonscissile 5'-end of the DNA for precise removal of the short 3'-overhang in L- and Y-structural DNA or to wedge into the double-stranded region for further digestion along the duplex. Biochemical assays were also conducted to demonstrate that TREX1 can indeed degrade double-stranded DNA (dsDNA) to a full extent. Overall, this study provided unprecedented knowledge at the molecular level on the enzymatic substrate processing involved in prevention of immune activation and in responses to genotoxic stresses. For example, Arg128, whose mutation in TREX1 was linked to a disease state, were shown to exhibit consistent interaction patterns with the nonscissile strand in all of the structures we solved. Such structure basis is expected to play an indispensable role in elucidating the functional activities of TREX1 at the cellular level and in vivo.