Effects of cations on small fragment of DNA polymerase I using a novel FRET assay.

Guojie Zhao, Suming Tang, Jun Li, Tianyu Hu, Yifu Guan

Acta biochimica et biophysica Sinica 2014 Aug

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DNA polymerase I (PolI) digested by protease produces a small fragment (SF) containing 5'-3' exonuclease activity. The 5'-3' exonuclease activity of polI cleaves the downstream RNA primer strands during DNA replication in vivo. Previous in vitro studies suggested its capability of cleaving duplex from 5' terminal and a flap-structure-specific endonuclease activity. From the crystal structures of other nucleases and biochemical data, a two-metal-ion mechanism has been proposed but has not been determined. In this study, we cloned, expressed, and purified the SF protein, and established a novel fluorescence resonance energy transfer (FRET) assay to analyze the catalytic activity of the SF protein. The effects of several metal ions on its catalytic capability were analyzed using this FRET assay. Results showed that Mg2+, Mn2+, and Zn2+ were able to activate the cleavage of SF, while Ca2+, Ni2 +, and Co2+ were not suitable for SF catalysis. The effects of K+, Na+, and dNTP were also determined.

Citation

Guojie Zhao, Suming Tang, Jun Li, Tianyu Hu, Yifu Guan. Effects of cations on small fragment of DNA polymerase I using a novel FRET assay. Acta biochimica et biophysica Sinica. 2014 Aug;46(8):659-67