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DNA Polymerase Ⅰ : Two distinct enzymes in one polypeptide chain

초록/요약

I studied structural function of DNA polymerase I. DNA polymerase I is composed of polymerase domain including proof-reading function, and 5'nuclease domain. Although the polymerase domain known as Klenow fragment shares common features with other polymerases, 5'nuclease domain is unique to eubacterial polymerase I family. In 1972, Setlow and Kornberg raised a question of why two distinct enzymes of polymerase and 5'nuclease are merged in one polypeptide. However, they could not find any difference between the mixture of two separated fragments and the intact whole DNA polymerase I. In 1995, Kim et. al. reported crystal structure of thermus aquaticus DNA polymerase including 5'nuclease domain, which is a member of polymerase I family. However, their structural analysis could not explain the cooperation of two domains. In 2000, Xu et. al. revisited this problem with advanced molecular biology. Unfortunately, they reached the same conclusion Setlow and Kornberg had arrived in 1972. Here we present an answer to this long-standing question by single molecule observation and mass spectrometric analysis. Single molecule DNA analysis shows that unleashed 5'nucleases cause severe DNA breaks more than whole DNA polymerase I. Furthermore, we present additional molecular reason by using mass spectrometric assay that unleashed 5'nuclease not only increases the life-time of single-stranded states, but also reduces polymerase processivity due to widened gaps, which raises the probability of DNA breaks.

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초록/요약

문헌조사를 통해 DNA Polymerase I 을 이용한 실험이 Enzyme 특성상 문제가 있을 수 있음을 인식하고 DNA Polymerase I 에서 하나의 Polypeptide Chain으로 연결된 Polymerase domain과 5'Nuclease domain을 따로 얻어서 DNA에 처리하고 MALDI TOF MS로 분석하였다. DNA Structure에 따라 Polymerase에 의한 dNTP의 incorporation 능력이 달라지는 것을 확인하고 Single Molecule DNA analysis를 통해서 두 domain이 붙어있는 것이 대장균 세포 입장에서 생존에 치명적인 DNA 손상을 감소시키는 결과를 불러온다는 실험적 근거를 제시하였다.

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