細胞遭受UV、環境中不同因子的壓力亦或是其他傷害時，常會造成DNA模板損傷而使得DNA複製過程中斷。在細菌中，最主要的DNA複製重啟機制是透過PriA所調控的途徑，此外還包含其他引物合成體(primosome)成員。然而，格蘭氏陰性菌與陽性菌之引物合成體成員除PriA與複製解旋酶外，其他成員皆有所不同。在格蘭氏陽性菌中其引物合成體成員包含：PriA，DnaD，DnaB與DnaC/I複合體，而且DnaD與DnaB在整個聚合得過程中，先於複製解旋酶進入複製重啟的位置。其中， PriA為一3端至5端之superfamily 2 DNA解旋酶，在DNA複製重啟過程中扮演兩個主要角色，PriA會辨識損傷之DNA並與其結合，同時協調幫助其它引物合成體 (primosome) 成員結合，使得DNA複製重啟過程得以順利完成。在本研究中，我們藉由生化實驗分別探討嗜熱脂肪芽孢桿菌PriA (GstPriA) 和肺炎鏈球菌PriA (SpPriA) 其ATP水解活性與DNA解旋能力。為了更進一步了解GstPriA和SpPriA之活性我們設計了不同型態之DNA受質進行實驗，實驗結果顯示不同於GstPriA之DNA解旋活性，SpPriA無法解旋高GC含量之DNA受質。然而，引物合成體成員DnaD則能夠做為輔助蛋白之角色 (accessory protein)提高PriA之酵素活性，並藉其結合單股DNA之能力使得停滯之複製叉不穩定幫助SpPriA克服其DNA解旋能力之限制。

When cells are stressed by ultraviolet irradiation or other environmental damaging treatments, DNA replication forks often encounter template DNA lesions, which can stall the progression of the replication forks. In bacteria, the PriA-dependent pathway is the major replication restart mechanism and it requires several primosome proteins. However, PriA in Gram-negative and –positive bacteria differ in their recruitment of subsequent loader proteins before recruitment of the replicative helicase. In Gram-positive bacteria, the primosome assembly process requires members PriA, DnaD, DnaB, DnaC/I complex. Moreover, DnaD and DnaB are known to have prime role in the assembly of helicase-loader complex. Among them, the PriA protein—a 3′ to 5′ superfamily-2 DNA helicase—is the key factor recognizing DNA lesions and it further recruits other proteins. Here, we investigated the PriA ATPase and helicase activities of Geobacillus stearothermophilus (GstPriA) and Streptococcus pneumoniae (SpPriA) through biochemical and kinetic analyses. Using various DNA duplexes with unstructured single-stranded regions, we observed distinct unwinding abilities for GstPriA and SpPriA according to increased GC content of duplex DNA substrate. In contrast to the unwinding activity of GstPriA helicase, we found that SpPriA is unable to unwind duplex DNA substrate with high GC content. However, the presence of DnaD loader protein allows SpPriA to unwind such duplex DNA. Our findings suggest that SpPriA possesses limited unwinding ability in high GC DNA and that DnaD acts as an accessory protein to destabilize stalled replication forks through its single-stranded DNA binding ability, thereby enhancing PriA unwinding activity.

摘要 I
Abstract II
致謝 IV
CONTENTS V
List of Table VIII
List of Figure IX
Chapter 1. Introduction 1
CHAPTER 2. MATERIALS AND METHODS
2.1 Plasmid construction 6
2.2 Purification of GstPriA and SpPriA 6
2.3 Purification of GstDnaB 8
2.4 Purification of SaDnaD and SpDnaD 9
2.5 Construction of DNA substrates 9
2.6 ATPase assay 10
2.7 Helicase activity assays 11
2.8 Equilibrium DNA binding assays 12
2.9 GST pulldown assays 13
2.10 Sedimentation-velocity analytical ultracentrifugation 13
2.11 Electrophoresis mobility shift assays 14
2.12 Protein homology modeling and quality assessment 15
2.13 Crystallizaiton 16
CHAPTER 3. RESULTS
3.1 Sequence disparity of PriA between Gram-negative and -positive bacteria 18
3.2 PriA ATPase activity 19
3.3 GstPriA and SpPriA reveal different helicase activities 20
3.4 DNA-binding activity of SpPriA and GstPriA 22
3.5 DnaB loader protein interacts physically with PriA and binds ssDNA substrate through its C-terminal domain 24
3.6 DnaD enhances the ATPase activity of PriA 27
3.7 PriA requires the co-loader DnaD to facilitate duplex DNA unwinding efficiency 30
3.8 The possibility for distinctive GstPriA and SpPriA helicase activities 31
CHAPTER 4. DISSCUSSION 35
CHAPTER 5. FUTURE WORK 39
TABLES 42
FIGRUES 45
APPENDIX 77
REFERENCES 84

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