Study on catalytic properties of flap structure : specific endonuclease 1 and establishment of new platforms for screening its inhibitors
Date of Issue2016-07-18
School of Physical and Mathematical Sciences
Flap structure-specific endonuclease 1 (FEN 1) is known to be essential for reproduction and development of eukaryotic cells. During DNA replication in cell division, a 5' flap structure is generated by polymerase 8. FENl subsequently emerges, specifically recognizes the produced flap structure, and further cleaves the phosphodiester bond. However, recent studies reveal few systematic results between catalytic property of FENl and different structures of DNA substrates that could emerge during replication. Here, we report the correlations between DNA-cleaving properties of FEN 1 and oligonucleotide substrates with various length of 5' flap or double strands. Substrate possessed 5' flap composed of 3 nucleotides and double strands composed of 16 nucleotides demonstrated high activity towards FENl catalysis. Since the enzyme FENl plays vital role in over-expression of DNA in cancer cells, FEN1 might be a sound target in certain anticancer treatment. Therefore we investigated effect of several small molecules on FENl catslysis, including choloroquine, ethidium bromide, netropsin, spermidine and cisplatin as well as inhibitory effect of phosphorothioate modified oligonucleotide substrates. Phosphorothioate bond was designed and modified at or adjacent to FEN 1 cleavage site, enzyme kinetics of FEN 1 using phosphorothioate-substituted oligonucleotides with modification at different positions was explored. The result showed that phosphorothioate modification at FENI cleavage site inhibited catalysis most efficiently. Based on activity study and inhibitory study, FENl oligonucleotide inhibitor was designed and its inhibitory effect was investigated. In addition, we have established two simple and efficient fluorescence platforms for screening FEN 1 inhibitors in vitro. The two fluorescence platforms developed were Cy3-(BHQ-2) donor-quencher fluorogenic platform as well as 2-aminopurine (2-AP) platform. The efficiency of FENI inhibitors could be measured by decrease of fluorescence emitted by Cy3 or 2-aminopurine. The Cy3-(BHQ-2) was more efficient than 2-AP platform; however 2-AP system was still significant to be utilized as validation by a different signal output for newly-discovered FENI inhibitors. Three organic inhibitors including PTPD, NSC-13755, aurintricarboxylic acid and our newly designed oligonucleotide inhibitor had been tested by fluorescence platforms and demonstrated a strong concentration-dependent inhibition. By using Cy3-(BHQ-2) fluoresence platform and 2-aminopurine fluorescence platform, IC5o value of organic inhibitors and oligonucleotide inhibitor was tested. Both platforms have demonstrated great potential to be further developed for screening FENI inhibitors. Besides, FENI activity using triplex DNA, RNA modified substrates at 5' flap structure were also explored. Flap-structured plasmid DNA was designed and synthesized for future study on FEN 1.