Browsing by Author "Yuan, Hanna S."

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Aromatic residues in RNase T stack with nucleobases to guide the sequence-specific recognition and cleavage of nucleic acids
(2015-12-01)
Duh, Yulander
;
Hsiao, Yu-Yuan
;
Li, Chia-Lung
;
Huang, Jason C.
;
Yuan, Hanna S.
RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3\'-to-5\' exonuclease activity is blocked by a 3\'-terminal dinucleotide CC in digesting both single-stranded RNA and DNA. Our previous crystal structure analysis of RNase T-DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3\'-terminal nucleobases. To elucidate if the pi-pi stacking interactions between aromatic residues and nucleobases play a critical role in sequence-specific protein-nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). The Escherichia coli strains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild-type RNase T in vivo. DNA digestion assays revealed W mutants shared similar sequence specificity with wild-type RNase T. However, the Y mutants exhibited altered sequence-dependent activity, digesting ssDNA with both 3\'-end CC and GG sequences. Moreover, the W and Y mutants had reduced DNA-binding activity and lower thermal stability as compared to wild-type RNase T. Taken together, our results suggest that the four phenylalanine residues in RNase T not only play critical roles in sequence-specific recognition, but also in overall protein stability. Our results provide the first evidence showing that the pi-pi stacking interactions between nucleobases and protein aromatic residues may guide the sequence-specific activity for DNA and RNA enzymes.
Aromatic residues in RNase T stack with nucleobases to guide the sequence-specific recognition and cleavage of nucleic acids
(2015-12)
Duh, Yulander
;
Hsiao, Yu-Yuan
;
Li, Chia-Lung
;
Huang, Jason C.
;
Yuan, Hanna S.
RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3\'-to-5\' exonuclease activity is blocked by a 3\'-terminal dinucleotide CC in digesting both single-stranded RNA and DNA. Our previous crystal structure analysis of RNase T-DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3\'-terminal nucleobases. To elucidate if the pi-pi stacking interactions between aromatic residues and nucleobases play a critical role in sequence-specific protein-nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). The Escherichia coli strains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild-type RNase T in vivo. DNA digestion assays revealed W mutants shared similar sequence specificity with wild-type RNase T. However, the Y mutants exhibited altered sequence-dependent activity, digesting ssDNA with both 3\'-end CC and GG sequences. Moreover, the W and Y mutants had reduced DNA-binding activity and lower thermal stability as compared to wild-type RNase T. Taken together, our results suggest that the four phenylalanine residues in RNase T not only play critical roles in sequence-specific recognition, but also in overall protein stability. Our results provide the first evidence showing that the pi-pi stacking interactions between nucleobases and protein aromatic residues may guide the sequence-specific activity for DNA and RNA enzymes.
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 Insights Into DNA Repair by RNase T- An Exonuclease Processing 39 End of Structured DNA in Repair Pathways
(2014-03-01)
Hsiao, Yu-Yuan
;
Fang, Woei-Horng
;
Lee, Chia-Chia
;
Chen, Yi-Ping
;
Yuan, Hanna S.
DNA repair mechanisms are essential for preservation of genome integrity. However, it is not clear how DNA are selected and processed at broken ends by exonucleases during repair pathways. Here we show that the DnaQ-like exonuclease RNase T is critical for Escherichia coli resistance to various DNA-damaging agents and UV radiation. RNase T specifically trims the 3 end of structured DNA, including bulge, bubble, and Y-structured DNA, and it can work with Endonuclease V to restore the deaminated base in an inosine-containing heteroduplex DNA. Crystal structure analyses further reveal how RNase T recognizes the bulge DNA by inserting a phenylalanine into the bulge, and as a result the 3 end of blunt-end bulge DNA can be digested by RNase T. In contrast, the homodimeric RNase T interacts with the Y-structured DNA by a different binding mode via a single protomer so that the 3 overhang of the Y-structured DNA can be trimmed closely to the duplex region. Our data suggest that RNase T likely processes bulge and bubble DNA in the Endonuclease V-dependent DNA repair, whereas it processes Y-structured DNA in UV-induced and various other DNA repair pathways. This study thus provides mechanistic insights for RNase T and thousands of DnaQ-like exonucleases in DNA 3-end processing. Author Summary DNA repair relies on various enzymes, including exonucleases that bind and trim DNA at broken ends. However, we know little about how an exonuclease precisely selects and trims a DNA broken end in specific repair pathways. In this study, the enzyme RNase T, previously known for its involvement in processing RNA substrates, is shown to also possess DNase activity. RNase T is a DnaQ-like exonuclease and is characterized in this work as the exoDNase responsible for trimming the 3 ends of structured DNA in various DNA repair pathways. Based on the high-resolution crystal structures of RNase T-DNA complexes, an insightful working model is provided showing how RNase T processes bulge, bubble, and Y- structured DNA in various DNA repair pathways. RNase T thus represents a unique structure-specific exonuclease with multiple functions not only in processing 3 overhangs of duplex RNA during RNA maturation, but also in processing 3 ends of bubble, bulge, and Y-structured DNA during DNA repair. These findings advance our understanding of the precise function of an exonuclease in DNA repair and suggest possible roles for thousands of members of DnaQ superfamily exonucleases in DNA repair and replication.
Structure and function of TatD exonuclease in DNA repair
(2014-09-15)
Chen, Yi-Chen
;
Li, Chia-Lung
;
Hsiao, Yu-Yuan
;
Duh, Yulander
;
Yuan, Hanna S.
TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life. It has been suggested that TatD participates in DNA fragmentation during apoptosis in eukaryotic cells. However, the cellular functions and biochemical properties of TatD in bacterial and non-apoptotic eukaryotic cells remain elusive. Here we show that Escherichia coli TatD is a Mg2+-dependent 3\'-5\' exonuclease that prefers to digest single-stranded DNA and RNA. TatD-knockout cells are less resistant to the DNA damaging agent hydrogen peroxide, and TatD can remove damaged deaminated nucleotides from a DNA chain, suggesting that it may play a role in the H2O2-induced DNA repair. The crystal structure of the apo-form TatD and TatD bound to a single-stranded three-nucleotide DNA was determined by X-ray diffraction methods at a resolution of 2.0 and 2.9 angstrom, respectively. TatD has a TIM-barrel fold and the single-stranded DNA is bound at the loop region on the top of the barrel. Mutational studies further identify important conserved metal ion-binding and catalytic residues in the TatD active site for DNA hydrolysis. We thus conclude that TatD is a new class of TIM-barrel 3\'-5\' exonuclease that not only degrades chromosomal DNA during apoptosis but also processes single-stranded DNA during DNA repair.