The mechanism of gap creation by a multifunctional nuclease during base excision repair.

During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3' end is released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes the enzymatic functions.

A ginsenoside-Rh1, a component of ginseng saponin, activates estrogen receptor in human breast carcinoma MCF-7 cells.

We have examined the possibility that a component of Panax ginseng, ginsenoside-Rh1, acts by binding to steroid hormone receptors such as receptors for estrogen, glucocorticoid, androgen, and retinoic acid. Ginsenoside-Rh1 activated the transcription of the estrogen-responsive luciferase reporter gene in MCF-7 breast cancer cells at a concentration of 50 microM. Activation was inhibited by the specific estrogen receptor antagonist ICI 182,780, indicating that the estrogenic effect of ginsenoside-Rh1 is estrogen receptor dependent. Ginsenoside-Rh1 induction of luciferase activity was dose-dependent in CV-1 cells transiently transfected with estrogen receptor and reporter plasmids. Next, we evaluated the ability of ginsenoside-Rh1 to induce the estrogen-responsive genes in MCF-7 cells. Ginsenoside-Rh1 increased c-fos and pS2 at the mRNA levels at 24h after treatment, although the effects were not as prominent as 17beta-estradiol. Western blot analysis showed that progesterone receptor protein was induced at 24h of treatment of ginsenoside-Rh1. However, ginsenoside-Rh1 failed to activate the glucocorticoid receptor, the androgen receptor, or the retinoic acid receptor in CV-1 cells transiently transfected with the corresponding steroid hormone receptors and hormone responsive reporter plasmids. These data support our hypothesis that ginsenoside-Rh1 acts as a weak phytoestrogen, presumably by binding and activating the estrogen receptor.