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 po...
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.