Identification of Transcription Factor AP-1 Bound to Untranslated 5′ Regulatory Region DNA of mdr1 Gene with Atomic Force Microscopy.

Multidrug resistance(MDR) phenotype of cancer cells is a major obstacle for cancer chemotherapy, this phenotype is main due to the overexprssion of the mdr1 gene. Transcription factor AP-1, which is one of important regulate proteins in the promoter region of mdr1 gene. To date, no direct data of AP-1-DNA regulating complexes on mdr1 gene. A novel method for identifying DNA-binding proteins from image analysis using atomic force microscopy(AFM) was developed. This study is to image and map the structure of Untranslated 5′ regulatory region DNA of mdr1gene, identificate and analysis of transcription factor AP-1 bound to Untranslated 5′ regulatory region DNA of mdr1 gene complex with atomic force microscopy, to find out the molecule mechanism of multidrug resistance.

Human leukemia adriamycin resistant strain K562/A02 was used as a target cells, transcription factor AP-1 was used as a target protein. Cultivate and PCR technique was used to amplify K562/A02 cells with the 769bp 5′regulatory region DNA of mdr1 gene, which fragment from −755 to +14. The amplified DNA products were purified by the PCR product purification kit, AP-1 of hela nuclear extract were purified by Sephadex spin-column filled with a bed of Sephadex G-100. The target DNA fragments were incubated with the target protein AP-1 at a 1:2 molar ratio in binding buffer and then immobilized the the AP-1-DNA complexes on a freshly cleaved mica surface which treated by MgCl₂. AFM was used to idificate image of the structure of target DNA and the AP-1-DNA complexes.

We show here the applicability of AFM in the quantitative analysis of the molecular mechanisms of DNA-protein interaction:

1. The optimum DNA concentration to yield well-absorbed DNA molecular on the mica surface was found to be 10ng/ul.

2. the contour of target DNA AFM image :the length of the DNA fragment measured by AFM image was 260.13± 2.29nm, the width was 11.88 ± 0.92nm, the mean height was 1.2nm(mean±SD, N = 50).

3. Sephadex spin-column (Ultrafree- MC 0.22) can be used to purificate DNA and protein-DNA complex, the contour data of AP-1 protein AFM image:: the width of protein_large was 30±3.2nm, protein _small was 19±2.8nm; the height of protein_large was 3.8±1.4nm, protein_small was 3.2±1.8nm (mean±SD, N = 30).

4. we got the contour data of pro- tein-DNA complexes: the width of protein _large was 28±2.7nm, the height was 3.4± 0.94nm (mean±SD, N = 8), the width of protein_small was 18±1.7nm, the height was 3.1±2.2nm (mean±SD, N = 22), and deduced the possible AP-1 site on mdr1DNA located between bases −126 and −115bp, this result is in close agreement with the expected −121 and −115 bp values.

the overall connection efficiency of protein was about 10%. The AFM method to visualize individual biomolecules allows us to investigate the conformation of protein-DNA complexes.