Suming Tang,
Jun Li,
Tianyu Hu and
Yifu Guan*
Abstract 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.
Keywords DNA polymerase I; FRET; exonuclease; cation
Received 2013-12-12
Accepted 2014-4-18
Funding This work was supported by the grants from the National Natural Science Foundation of China (31070705, 81371896, and 81301517) and the Doctoral Startup Found of Liaoning Province (20131139).
* Correspondence address Tel/Fax: +86-24-23255240; E-mail: [email protected]