Role of AP endonuclease in the repair of C1'-oxidized abasic site in Escherichia coli = Escherichia coli 에서 C1’-oxidized abasic site의 수리에 AP endonuclease가 하는 역할

국문 초록 (Abstract)

Oxidized abasic site는 DNA에서 발생하는 다양한 산화적 손상들 중 하나로 특히 항암제나 UV 등에 의해 1번 탄소에 oxidation이 일어나면 특이적인 C1’-oxidized abasic site, 즉 2-deoxyribonolactone (dL) 이 발생하게 된다. 이렇게 손상된 DNA를 수리하는 세포 수리 기작으로는 base excision repair (BER) 가 있는데, 최근에 dL과 다양한 BER enzyme 간에 자발적인 DNA-protein cross-link (DPC) 가 생성됨이 밝혀졌다. 특히mammalian BER 기작에서 중요한 역할을 하는 Polβ와 dL-DNA간에 DPC가 생성되는 것이 밝혀졌으며 (Sung et al, 2005) E. coli bi-functional DNA glycosylases인 Fapy-DNA glycosylase (Fpg) 와 endonuclease III (Endo III) 가 DNA의 절단 없이 dL과 DPC를 형성하는 것이 밝혀졌다 (Saparbaev et al, 2002; Kroeger et al, 2003). 본 논문에서는, BER을 구성하는 중요한 enzyme들 중 하나인 AP endonuclease의 유무에 따른 DPC 형성의 pattern을 관찰하기 위해, gene cloning을 통하여 특정 E. coli 균주에 BER을 구성하는 특정 enzyme들을 overexpression시켰다. 그리고 각 균주의 cell extract를 만들고 dL-DNA와 반응시킨 후 SDS-PAGE를 통하여 분석하였다. 그 결과, 특정 E. coli protein과 dL-DNA 간에 자발적인 DPC가 형성되며 이는 AP endonuclease의 유무에 상관없이 나타나는 것이 밝혀졌다. 또한 Fpg와 Endo III가 AP endonuclease와 함께 작용하여 DPC의 형성을 억제하였다. 이는 Fpg와 Endo III가 AP lyase activity를 통해 DNA를 절단함으로써 BER을 통하여 더욱 효과적으로 dL을 수리하는 것으로 사료된다. 또한, DPC를 형성하는 E. coli protein과 mammalian Polβ의 dL에 대한 우선적인 binding을 확인하기 위하여 같은 방법으로 실험을 진행하였다. 그 결과, Polβ에 의한 DPC 형성은 E. coli cell extract와 함께 incubation될 때 억제되었으며, purified Endo IV를 AP endonuclease가 deletion된 E. coli BW528 균주의 extract에 처리하여 dL의 5’을 절단해주자 E. coli protein과 생성되던 DPC가 크게 감소하였다. 이는 E. coli protein에 AP sit의 3’을 절단하는 AP lyase activity가 포함되어 있으며 E. coli와 Polβ 간에 dL에 대한 경쟁적인 binding이 형성된다는 것을 말해준다. 또한 종합적으로 볼 때 E. coli와 mammalian cell 간에 발생하는 dL-DPC 형성 기작이 전혀 다른 것임을 말해주는 결과이다. 그러나 NCS를 통해 E. coli genome에 dL을 발생시키자 AP endonuclease가 존재하는 균주의 생존률이 가장 낮았으며 Fpg와 Endo III를 overexpression시킨 균주의 생존률이 크게 향상되었다. 이는 AP endonuclease가 BER을 통한 dL의 수리에 중요한 역할을 담당하는 mammalian cell의 기작과는 다르게, E. coli에서는 AP endonuclease로 인한 DNA를 절단이 오히려 세포에 독성으로 작용하며 AP lyase가 매개하는 BER에 의해 손상된 DNA가 수리되는 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

Oxidized abasic residues arise as a major class of DNA damage by variety of agents involving free radical attack and oxidation of deoxyribose sugar components. Especially, 2-deoxyribonolactone (dL) is a C1'-oxidized abasic site in DNA produced by various genotoxic agents including gamma-radiolysis and neocarzinostatin chromophore (NCS). Recently, it has been reported that the formation of covalent cross-link can form spontaneously between dL-DNA and E. coli bi-functional DNA glycosylases, fapy-DNA glycosylase (Fpg), and endonuclease III (Endo III), without DNA strand scission. We show here the inhibition of spontaneous cross-linking formation by AP lyase activity between dL-DNA and E. coli major proteins. Such inhibition by AP lyase activity increased more when proficient in AP endonuclease. These results are dependent on the repair function of AP lyase, and both AP lyase and AP endonuclease activities prevent the formation of DNA-protein cross-link (DPC). Also, recent study reported that the formation of covalent cross-link can occur spontaneously between dL-DNA and mammalian Polβ, but only after the incision of DNA by AP endonuclease at the site of dL. This paper shows here the first evidence of dL-mediated cross-linking (DPC) by E. coli protein that occurs independently in the presence of AP endonuclease activity and the formation of dL-mediated DPC decreased when the concentration of AP endonuclease increased. The formation of dL-mediated cross-link by Polβ was inhibited when the enzyme was co-incubated with cell extracts of both E. coli BW32 (xth+, nfo+) and BW528 (xth-, nfo-), which were deficient or proficient in major AP endonuclease activity, respectively. Such inhibition effected by BW32 extracts was caused by the competition between Polβ and E. coli major proteins involved in dL-mediated DPC. On the other hand, formation of DPC by Polβ was largely inhibited by BW528 extracts, whereas the rate of DPC formation mediated by E. coli major protein was not affected. These observations suggest that the major E. coli protein involved in dL-mediated cross-link contains AP lyase activity that mediates the incision of DNA at the 3' of dL site. The generation of dL lesions in E. coli genome by NCS was more toxic to the cells when the AP endonucleases were present. Our data show that typical DNA repair function of AP endonuclease results in deleterious effects in E. coli by producing DNA strand scissions rather than mediating DNA-protein cross-links when it encounters C1'-oxidized deoxyribose lesions in DNA.

목차 (Table of Contents)

• List of Abbreviations ⅳ
• List of Figures ⅴ-ⅵ
• List of Tables ⅶ
• Abstracts 1-3
• Ⅰ. Introduction = 4-16
• Ⅱ. Materials and Methods = 17-25
• 1. Materials = 17
• 2. Construction of expression vectors = 18-19
• 3. Transformation of E. coli = 19-20
• 4. Overexpression and purification of recombinant pKK-Fpg and pKK-Nth = 20-21
• 5. Overexpression of pKK-Nfo = 21-22
• 6. Analysis of dL-mediated DPC formation = 22-23
• 7. Polyacrylamide gel electrophoresis = 23-24
• 8. Cell viability assay = 25
• Ⅲ. Results = 26-36
• 1. Cloning of Endo IV, Fpg and Endo III = 26
• 2. Identification of cloning of pKK-Nfo with reduced AP site = 26-27
• 3. Identification of cloning of pKK-Fpg and pKK-Nth = 28
• 4. The presence of AP endonuclease prevents stable DNA-protein cross-link = 28-30
• 5. Identification of the cross-linking between DNA and protein = 30-31
• 6. Covalent cross-link formation between dL and E. coli JM109 transformed with pKK-Fpg cell-free extract proteins = 31-32
• 7. Covalent cross-link formation between dL and E. coli JM109 transformed with pKK-Nth cell-free extract proteins = 32-33
• 8. Competition analysis between Polβ and Endo IV = 33-34
• 9. AP endonuclease results in deleterious effects by producing dL lesions by NCS in E. coli = 34-36
• 10. AP lyase plays a key role in the cellular repair of dL lesions induced by NCS in E. coli = 36
• Ⅳ. Discussion = 37-42
• Ⅴ. Reference = 43-52
• Ⅵ. Figures = 53-84
• Ⅶ. Tables = 85-86
• Abstracts (in Korean) = 87-90