Enzymatic studies on nucleic acid binding protein dead box protein csha from staphylococcus aureus and rna dependent rna polymerases from viruses

Dissertation for Degree of Doctor Supervisor: Prof Dong-Eun Kim Enzymatic Studies on Nucleic Acid Binding Proteins: DEAD-box Protein CshA from Staphylococcus aureus and RNA-dependent RNA Polymerases from Viruses Submitted by NGUYEN THI DIEU HANH February, 2015 Department of Bioscience and Biotechnology Graduate School of Konkuk University Enzymatic Studies on Nucleic Acid Binding Proteins: DEAD-box Protein CshA from Staphylococcus aureus and RNA-dependent RNA Polymerases from Viruses A Dissertation submitted to the Department of Bioscience and Biotechnology and the Graduate School of Konkuk University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Submitted by NGUYEN THI DIEU HANH October, 2014 This certifies that the Dissertation of NGUYEN THI DIEU HANH is approved Approved by Examination Committee: Chairman Member Member Member Member November, 2014 Graduate School of Konkuk University TABLE OF CONTENTS List of Tables .iv List of Figures iii List of Scheme vii Abstract viii Chapter Characterization of DEAD-box protein CshA from Staphylococcus aureus on RNA substrates 1.1 Introduction 1.1.1 Staphylococcus aureus 1.1.2 DEAD-box protein 1.1.3 The role of DEAD-box protein CshA in S aureus 1.2 Materials and Methods 1.2.1 Preparation of recombinant S aureus CshA 1.2.2 Preparation of RNA oligonucleotides 1.2.3 RNA-dependent ATP hydrolysis 11 1.2.4 Duplex RNA unwinding assay 11 1.2.5 Ribonuclease assay 12 1.2.6 RNA strand annealing assay 13 1.2.7 RNA strand exchange assay 13 1.3 Results 15 1.3.1 Duplex RNA stimulates ATP hydrolysis by S aureus CshA 15 1.3.2 S aureus CshA is unable to catalyze RNA unwinding 17 1.3.3 S aureus CshA possesses ribonuclease activity 19 i 1.3.4 S aureus CshA is an endoribonuclease that cleaves ssRNA at preferred sequences 23 1.3.5 S aureus CshA possesses RNA strand annealing activity 30 1.4 Discussion 36 1.5 Conclusion 41 Chapter Functions of DEAD-box Protein CshA from Staphylococcus aureus on DNA substrates 42 2.1 Introduction 42 2.2 Methods 44 2.2.1 Nucleic acid substrates 44 2.2.2 DNA filter-binding assay 47 2.2.3 ATP hydrolysis 48 2.2.4 Duplex DNA unwinding assay 48 2.2.5 DNA strand exchange assay 49 2.2.6 DNA strand annealing assay 50 2.2.7 Inhibition assay of DNA strand annealing 51 2.3 Results 53 2.3.1 S aureus CshA binds to duplex DNA with overhangs 53 2.3.2 DNA strand exchange is promoted by S aureus CshA 57 2.3.3 S aureus CshA catalyzes strand annealing of complementary ssDNA into dsDNA 67 2.3.4 S aureus CshA anneals partial duplex DNAs into nicked or gapped dsDNA 74 2.3.5 CshA-mediated DNA strand annealing is inhibited by dsDNA and high NaCl concentration 78 ii 2.4 Discussion 82 2.5 Conclusion 87 Chapter Efficient colorimetric assay for RNA synthesis by viral RNA-dependent RNA polymerases using thermostable pyrophosphatase 88 3.1 Introduction 88 3.2 Materials and Methods 91 3.2.1 Preparation of recombinant proteins 91 3.2.2 Pyrophosphate hydrolysis assay 91 3.2.3 Colorimetric assay of RdRp activity 92 3.2.4 Radioactive assay of RdRp activity 92 3.2.5 Inhibition assay of DMUT (3'-deoxy 5-methyl-uridine-5'triphosphate) on RdRp activity 93 3.3 Results and Discussion 93 3.3.1 Conversion pyrophosphate to inorganicphosphate by thermostable pyrophoaphatase 93 3.3.2 Comparison of the colorimetric assays to measure RdRp activity 96 3.3.3 Use of the colorimetric assay to detect RdRp activity from the crude cellular extract 97 3.3.4 Application of the colorimetric assay for RNA synthesis by RdRp to screen inhibitor 101 3.4 Conclusions 103 References 104 iii List of Tables Table 1.1 Sequences of single-stranded RNAs used in this study 10 Table 1.2 Structures of double-stranded RNA substrates used in this study 10 Table 2.1 Oligonucleotides used in this study 45 Table 2.2 Structures of DNA substrates 46 iv List of Figure Figure 1.1 Scheme of different sites of staphylococcal infections and biofilm of Staphylococcus aureus Figure 1.2 Conversed motifs of DEAD-box proteins Figure 1.3 The current model of Bacillus subtilis and Staphylococcus aureus Figure 1.4 Model of CshA in the degradation of the agr mRNA in the wild type and CshA mutant type Figure 1.5 RNA-dependent ATP hydrolysis by S aureus CshA 16 Figure 1.6 RNA duplex unwinding by Staphylococcus aureus CshA 18 Figure 1.7 CshA degrades the single-stranded RNA region of duplex RNA substrates 21 Figure 1.8 CshA did not possess 5’-exoribonuclease activity 24 Figure 1.9 CshA is an endoribonuclease 26 Figure 1.10 Mapping of RNA cleavage sites on two different single-stranded RNA substrates (R0 and BR0) in RNA degradation by CshA 29 Figure 1.11 RNA strand annealing activity of CshA 32 Figure 1.12 S aureus CshA does not catalyze RNA strand exchange 35 Figure 1.13 Catalytic activities of S aureus CshA on RNA substrates 40 Figure 2.1 DNA binding and ATP hydrolysis by the recombinant CshA from S aureus 55 Figure 2.2 CshA shows DNA strand exchange activity rather than dsDNA unwinding 58 Figure 2.3 DNA strand exchange activity of CshA on various dsDNA substrates 63 v Figure 2.4 Assay for the DNA strand exchange activity of CshA with 3′-tail dsDNA substrate to form a forked dsDNA product 65 Figure 2.5 ssDNA strand annealing by CshA 69 Figure 2.6 ssDNA strand annealing activity of CshA to form diverse dsDNA products 72 Figure 2.7 Formation of duplex DNAs with gap and nick is promoted by CshA 76 Figure 2.8 Inhibition of CshA-catalyzed DNA strand annealing with dsDNA competitor and high concentration of salt 80 Figure 2.9 Plausible roles of DNA annealing and DNA strand exchange activities by CshA in dsDNA break repair 86 Figure 3.1 Colorimetric detection of RNA polymerase activity using thermostable pyrophosphatase 95 Figure 3.2 Comparison of RdRp activity assay methods between colorimetric assay and radioactive assay 97 Figure 3.3 The colorimetric assay of FMDV 3Dpol RdRp from the crude cellular extract 99 Figure 3.4 Inhibition of 3’-deoxy 5-methyl-uridine-5’triphosphate (DMUT) on the activity of FMDV 3Dpol 102 vi List of Scheme Scheme 3.1 Reaction scheme for colorimetric assay of RNA polymerase 90 vii cellular extract For a wide application of the colorimetric method, the colorimetric RdRp assay were also carried out with the crude cellular extract containing FMDV 3Dpol RdRp that was collected from the crude extracts of cells induced to express enzyme (Fig 3.3) Time-courses of inorganic phosphate accumulation with the crude cellular extract was compared with the one with the purified enzyme (Fig 3.3B) Although the phosphate background is relatively high with the crude extract as compared with the purified enzyme, the colorimetric assay provided a significant increase of absorbance due to net increase of phosphate amounts in the RNA synthesis reaction Comparison of RdRp activity assay methods between colorimetric assay and radioactive assay at the same protein amount (2.7g) showed that Pi accumulation in the reaction of colorimetric assay performed with the crude extract was 27.4 %, when the one produced with the purified RdRp was set to 100 %, whereas the RNA synthesized in the radioactive assay with the crude extract was 32.4 %, when the RNA produced with the purified RdRp was set to 100 %, which is close to the value obtained by the colorimetric assay method (Fig 3.3C) Thus, the current colorimetric assay is compatible with the crude cellular extracts containing the RNA polymerase despite of relatively low ratio of signal to background 98 Figure 3.3 The colorimetric assay of FMDV 3Dpol RdRp from the crude cellular extract (A) SDS-PAGE analysis of purified RdRp enzyme (denoted as purified enzyme in left panel) and crude cellular extract containing FMDV 3Dpol 99 enzyme (denoted as crude extract in right panel) The purified enzyme (10 g/lane) and the crude extract of E coli cell lysate (40 g/lane) after induction of the protein expression were subjected to SDS-PAGE (10 %) analysis, and the gels were stained with Coomassie Brilliant Blue (B) Time course of phosphate accumulation in the RNA polymerization reaction with purified enzyme or the crude cellular extract with or without RNA template The background of the polymerase reactions were carried out similarly in the absence of RNA template to determine the inorganic phosphate concentration, which was generated by other nucleotide hydrolyzing enzymes not RdRp polymerase (C) Comparison of RNA synthesis activity between the crude cellular extract and the purified FMDV RdRp after 90 of the RNA synthesis reaction, which was measured by the colorimetric assay and the radioactive assay The differences of inorganic phosphate concentration between the reactions in the presence and absence of RNA template in both reactions (panel B) were plotted as the RNA polymerase activity (inset graph) In the radioactive assay, the polymerization reactions were carried out similarly in the same reaction mixture as the colorimetric assay except of UTP spiked with -32P-labeled UTP (0.4 Ci/nmol) The RNA products after 90 of polymerization reaction were resolved on 10 % polyacrylamide gel (inset gel image) 100 3.3.4 Application of the colorimetric assay for RNA synthesis by RdRp to screen inhibitor The use of this colorimetric assay was further validated by evaluation of a known inhibitor against FMDV 3Dpol RdRp, which is a nucleotide derivative; 3'deoxy 5-methyl-uridine-5'triphosphate (DMUT; TriLink BioTech, San Diego, CA) [64] The colorimetric assay showed that FMDV 3Dpol activity was inhibited by DMUT with a decreased rate of 0.012 min-1 from 0.024 min-1 in the reaction without DMUT (Fig 3.4A) This result was consistent with observed rates of RNA synthesis, which was obtained by the radioactive assay; 0.012 min-1 and 0.033 min1 with and without DMUT, respectively (Fig 3.4B) In the presence of increasing concentrations of DMUT, FMDV 3Dpol activity was measured with the colorimetric assay FMDV 3Dpol was gradually inhibited as DMUT concentration increases, which was fit to a hyperbolic equation with an IC50 value of 37.3 M for DMUT concentration (Fig 3.4C) 101 Figure 3.4 Inhibition of 3’-deoxy 5-methyl-uridine-5’triphosphate (DMUT) on the activity of FMDV 3Dpol at various incubation time measured by the colorimetric assay (top panel) and the radioactive assay (middle panel) and at varying concentrations of DMUT (bottom panel) RNA synthesis by M FMDV 3Dpol on 0.1 M poly-rA/dT18 was carried out in the presence and absence (designated as control) of 200 M DMUT for the designated time, or the reactions were performed at varying DMUT concentrations for h 102 3.4 Conclusions In summary, I developed a simple colorimetric assay to quantify the RNA synthesis activity of RdRp by quantifying inorganic phosphate produced by thermostable inorganic pyrophosphatase that hydrolyzes the released pyrophosphates during nascent RNA synthesis reaction Use of thermostable pyrophosphatase facilitates thermal quenching of RdRp reaction without changing the components of 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Proteins: DEAD- box Protein CshA from Staphylococcus aureus and RNA- dependent RNA Polymerases from Viruses Nguyen, Thi Dieu Hanh Department of Bioscience and Biotechnology Graduate School of Konkuk