SUNY at Albany
June 19-23, 2001
Structures and Characteristic of Telomeric DNA in Cells by Atomic Force Microscopy
Telomeres are specialized terminal elements in the eukaryotic nucleus, composed of tandem repeats of short DNA sequences and specific proteins. Some studies reported telomeric G-strand DNAs from a variety of organisms adopt compact structure, the most state of which is explained by the formation of G-quartets. Recently,Griffth and Lange showed by electron microscopy (EM) that telomeres end in a large duplex loops, but its structure characteristic has not been defined in detail, This force us to further investigate on telomeres structure in cells. In this paper, HeLa cells were treated with or without psoralen-UVA, and then DNA was cleaved with Rasl and Hinf, purified by Bio-Gel P-2 column. Purified telomeres DNA was directly observed by tapping model AFM. A circular loop with a tail structure in the purified psoralen cross-linked telomeric DNA species from HeLa cells was observed by AFM. According to its apparent height in AFM, the circular potion of loops is assumed to be double- (T-loop), but the loop-tail junction is assumed to be three-strand (D-loop) or few tetra-stranded (braid knot) structure, respectively. AFM images also showed that G-quartets were presented in the loops. However, for the cells without psoralen-UVA treatment, only few similar looped DNA can be observed occasionally in crude cell DNA extract without deprotein, enzyme digest and purification. No any looped DNA with a tail was presented after deprotein, suggesting that deproteinization caused the change of telomeric DNA, suggesting the psoralen cross-linking of the DNA strands is predicted to preserve the T-loops after removal of protein (mainly TRF2). It has proved that intrastrand fold-back DNA tetra-stranded specie with telomere sequences presented in the cell DNA extract digested by DNase I, and K+ can stabilize the structure. Our results also showed that up-regulated protein by psoralen is 1.8 fold higher than that of control and induction of p53 mRNA was in the 1.5-2.0-fold range. It may be related to inhibition of TRF2, which led to the changes of the p53 gene expression. AFM results indicated that telomeres in human cells may end in double stranded T-loop and three-stranded D-loop by the single-stranded overhang is tucked back inside the double-stranded DNA or a four stranded braid knot by interaction between the double-strands, and that the G-rich strand in telomere DNA can adopted a four stranded G-quartets by intrastrand fold-back. Above results can explain the different results from single G stranded DNA in vitro and telomeric DNA in cells. Which is advantage in the further studies on structure and function of telomere in cells.
En-Hua Cao (1), Ai Chen (1), Xian-Yan Zhang (1), Jing-Fen Qin (1), Da-Ge Liu (2), Chen Wang (2), and Chun-li Bai (2)
(1) Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 China (2) Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China