Book of Abstracts: Albany 2003
June 17-21 2003
Assembly of p53 Protein with T-loop Junction in Telomeres and Apoptosis
Some investigations have implicated p53 protein has a strong affinity for ss telomeric overhang of the telomere and tightly binds Holliday junctions in vitro. These data suggest that the tumor suppressor protein p53 might be involved directly in the telomere structure state in cells.
Our recent studies showed in human gastric cancer MGC-803 cells, pretreatment of As2O3 induced the changes of telomere lengths: some telomeres was longer, whereas others were very short or lost entirely, resulted in the telomere-telomere fusions. In same time, increase of p53 expression and cell apoptosis also was observed. Addition of DODC (diethyloxadicarbocyanine), an inductor of hairpin G-quadruplex, could synergistically enhance the apoptosis induced by arsenic, thought it alone could not induce apoptosis. It is possible that the increase of senescence to arsenic might be related directly to p53-telomere DNA interaction in cells. The telomere state altered signals p53 directly through association with the t-loop junction.
Using atomic force microscopy (AFM), we observed that some looped structures existed in crude cell extract, but it disappear in the deproteinized sample. However, the large loops with a tail structures were observed in the purified telomeric DNA species by UV cross-linking. The circular portion of loops is assumed to be double-strand structure according to its apparent height, to be triplex or tetraplex structure for the loop-tail junction. It further evidenced that mammalian telomeres end in a large duplex loop structure with a tail (t-loops) in cells. Further studies showed the triplex structure consists of a 3`ss overhang of TTAGGG repeat into to the duplex TTAGGG portion of the telomere to generate a D-loop in the present of TRF2 alone. No formation of t-loop was observed in the present of p53 alone, but p53 binding to the 3' ss overhang was observed. The tetraplex structure might be the C-rich strand of the ss/ds telomeric junction may also invade the duplex, resulting in the formation of a Holliday junction-like structure in the present of both TRF2 and p53. TRF2-mediated t-loop formation was increased by the addition of p53 due to the formation of a Holliday junction-like structure. The formation of t-loops decreased by the addition of DODC due to formation of hairpin G-quadruplex structure in 3`ss overhang. These results suggest that t-loop structure change (capped or uncapped state) at the telomeres may signals p53 directly, then to trigger apoptosis through the p53/ATM-dependent DNA damage checkpoint pathway.
This work was supported by a grant from National Science Foundation of China
1Institute of Biophysics