Biochemical characterization of enzyme activities of Hrq1 and eIF1A proteins

다국어 초록 (Multilingual Abstract)

RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolisms for maintenance of genome stability. Five RecQ helicases are found in human, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here I show that 3’-5’ helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. I also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3’-tail (70-nt) for efficient unwinding of duplex DNA. Hrq1 was also found to have potent DNA strand annealing activity.
Hrq1 displayed structure-specific DNA-binding property. It preferentially bound to bubble and displacement-loop structures. In addition, it was able to resolve displacement-loop structures but hardly unwound bubble DNA. The resolution of displacement-loop was strongly stimulated by the presence of 3’-tail. Consistent with a recent report that human RECQL4 localizes at telomere, Hrq1 was found to more tightly bind to yeast telomeric sequences than random sequences. Moreover, DNA substrate with telomeric sequence was more susceptible to unwinding reaction by Hrq1. These results indicate that Hrq1 is able to function in various DNA metabolisms for maintenance of genome stability that deserves further characterization to expand our understanding of RecQ helicases.

다국어 초록 (Multilingual Abstract)

Translation initiation factor eIF1A is highly conserved among all eukaryotes, and performs essential functions in the formation of 43S preinitiation complex, and mRNA scanning. In this study, I found that an RNA annealing activity is intrinsically associated with eIF1A. Schizosaccharomyces pombe, Saccharomyces cerevisiae, and human eIF1As were isolated in their recombinant forms in order to determine their RNA annealing activities. A truncated eIF1A devoid of both N- and C-terminal domains proved most active, indicating that the activity is localized in the OB-fold domain. Some N- or C-terminal His tag fusions were shown to make the proteins inactive. This is probably caused by shielding of the RNA binding surface, as the proteins were activated via partial proteolytic digestion. I also found that eIF1A formed a stable complex with a short double-stranded RNA in gel mobility shift assays.
To determine the active sites for the biochemical activities of eIF1A and to investigate whether they are essential for yeast cell growth, various mutations were introduced in TIF11, the eIF1A-encoding gene, and the resulting mutant proteins were purified. All point mutations in the OB-fold domain, except R57D, impaired both RNA annealing and dsRNA binding activities, indicating that the intact OB-fold domain is required for both activities. Viabilities of the mutant yeast cells were not correlated with RNA annealing activity. In the case of R57D and K94D, the instabilities of these mutant proteins most probably give rise to inviability of the mutant cells. In conclusion, these results indicate that eIF1A may function as an RNA chaperone, inducing conformational changes in rRNA in the 43S preinitiation complex.

목차 (Table of Contents)

• PART 1: Biochemical characterization of Hrq1 helicase 1
• ABSTRACT 2
• INTRODUCTION 4
• MATERIALS AND METHODS 12
• Oligonucleotides and DNA substrate preparation 12
• Cloning and purification 12
• Helicase assay 13
• DNA strand annealing 14
• DNA-binding assay 15
• DNA-dependent ATPase activity of Hrq1 15
• RESULTS 19
• Purified Hrq1 protein contains DNA helicase activity 19
• Hrq1 is a 3-5 helicase and its activity is stimulated by fork structures 22
• Hrq1 requires a long 3-tail length for helicase activity 25
• Hrq1 has both processive helicase and DNA strand annealing activities 31
• Hrq1 displays DNA binding preference for bubble structures 34
• Hrq1 hardly unwinds bubble structures 36
• Hrq1 preferentially binds and unwinds D-loop structure 40
• Hrq1 displaces the invading strand from D-loop without opening it 47
• Sequence preferences for DNA binding and ATPase activities of Hrq1 51
• DISCUSSION 59
• REFERENCES 62
• PART 2: Biochemical characterization of eIF1A 66
• ABSTRACT 67
• INTRODUCTION 68
• MATERIALS AND METHODS 73
• Purification of the native eIF1A from S. pombe 73
• Oligonucleotides and RNA substrate preparation 73
• RNA annealing and gel mobility shift assays 74
• Cloning and purification of eIF1As and IF1 75
• Cloning of TIF11 mutants plasmids 76
• Construction of yeast strains 76
• RESULTS 79
• S. pombe eIF1A shows an RNA annealing activity 79
• Yeast eIF1A also contains RNA annealing activity in its OB-fold domain 79
• Activation by proteolytic digestion demonstrates that the RNA annealing activity is intrinsic to eIF1A 85
• eIF1A binds to dsRNA 87
• The eIF1A proteins are defective in RNA annealing activity 89
• Effect of tif11 mutations on the yeast cell growth 94
• DISCUSSION 103
• REFERENCES 107
• Part Ⅰ 16
• Table 1. Oligonucleotides used in this study 16
• Table 2. Preference of DNA-binding and ATPase activities of Hrq1 54
• Part Ⅱ 78
• Table 1. Primers for in vitro mutagenesis 78
• Table 2. Summary of biochemical properties of mutant eIF1As 101
• Part Ⅰ 5
• Figure. 1-1. The RecQ helicase play multiple roles in the maintenance of genome stability 5
• Figure. 1-2. The RecQ helicase family 7
• Figure. 1-3. DNA substrates of RecQ family helicases 9
• Figure. 1-4. Schematic structures of DNA substrates used in helicase and gel shift assays of Hrq1 18
• Figure. 1-5. Co-migration of helicase and DNA-dependent ATPase activities of purified Hrq1 20
• Figure. 1-6. Purification and helicase activity of Hrq1 protein 21
• Figure. 1-7. Effect of various condition of ATP, Mg2+,pHs on the helicase activity 23
• Figure. 1-8. Polarity of DNA unwinding by Hrq1 24
• Figure. 1-9. Preference for a fork-structured substrate 26
• Figure. 1-10. Helicase activity on various DNA substrates 27
• Figure. 1-11. Stimulation of helicase activity by a long 30-tail 29
• Figure. 1-12. Hrq1 possesses helicase activities. 32
• Figure. 1-13. DNA strand annealing activity of Hrq1 33
• Figure. 1-14. Mg2+-dependent binding of Hrq1 35
• Figure. 1-15. Complex formation of Hrq1 with ss- or dsDNA and bubble substrates 37
• Figure. 1-16. Hrq1 possesses weak helicase and bubble DNA strand annealing activities for bubble structure 39
• Figure. 1-17. Dna2-cleavage of the 5-ssDNA ends generated by Hrq1 helicase 41
• Figure. 1-18. Hrq1 preferentially binds to bubble and D-loop DNA-binding protein 43
• Figure. 1-19. Hrq1 efficiently unwinds the 3-tailed D-loop substrate 45
• Figure. 1-20. D-loop strand annealing activity of Hrq1 46
• Figure. 1-21. The D-loop Substrate helicase assays of Hrq1 and Dna2 48
• Figure. 1-22. Hrq1 shows higher levels of DNA binding and DNA-dependent ATPase activities with telomeric and random sequence than with random sequence 49
• Figure. 1-23. Stimulation of Helicase activity by 3 tail telomeric sequence 50
• Figure. 1-24. Quantitation of DNA-dependent ATPase activity of Hrq1 differentially stimulated by various DNA sequence 52
• Figure. 1-25. Sequence specificity DNA binding activity of Hrq1 55
• Figure. 1-26. Sequence specificity DNA binding activity of Hrq1 56
• Figure. 1-27. Sequence specificity DNA binding activity of Hrq1 57
• Figure. 1-28. Hrq1 displays sequence-specific DNA binding activity Quantitative comparison of Hrq1 DNA binding activity to various DNA substrates 58
• Part Ⅱ 69
• Figure. 2-1. The Multi-function of RNA chaperones in RNA metabolism 69
• Figure. 2-2. Structure of eIF1A 71
• Figure. 2-3. Isolation of an RNA annealing activity from S. pombe 80
• Figure. 2-4. Co-migration of RNA annealing activity with the purified S. pombe eIF1A 81
• Figure. 2-5. Purification of eIF1As 83
• Figure. 2-6. Comparison of RNA annealing activities of various eIF1A preparations 84
• Figure. 2-7. Activation of eIF1As by partial proteolysis 86
• Figure. 2-8. Complex formation of eIF1A and dsRNA 88
• Figure. 2-9. Comparison of the eIF1A and IF1 amino acid sequences from different species 90
• Figure. 2-10. Purification of eIF1As 92
• Figure. 2-11. Comparison of RNA annealing activities of wild-type and mutant eIF1A preparations 93
• Figure. 2-12. Comparison of RNA annealing activities of wild-type and mutant eIF1A preparations eIF1A 95
• Figure. 2-13. Complex formation of eIF1A and dsRNA 96
• Figure. 2-14. Plasmid shuffling test of tif11 mutants with its own promoter 98
• Figure. 2-15. Western blot analysis of eIF1As 99
• SUMMARY IN KOREAN 109
• ACKNOWLEDGEMENTS 111
• CURRICULUM VITAE 112