Synthesis of novel poly(dG)-poly(dG)-poly(dC) triplex structure by Klenow exo- fragment of DNA polymerase I.
The extension of the G-strand of long (700 bp) poly(dG)-poly(dC) by the Klenow exo(-) fragment of DNA polymerase I yields a complete triplex structure of the H-DNA type. High-performance liquid chromatography analysis demonstrates that the length of the G-strand is doubled during the polymerase synthesis. Fluorescence resonance energy transfer analysis shows that the 5' ends of the G- and the C-strands, labeled with fluorescein and TAMRA, respectively, are positioned close to each other in the product of the synthesis. Atomic force microscopy morphology imaging shows that the synthesized structures lack single-stranded fragments and have approximately the same length as the parent 700 bp poly(dG)-poly(dC). CD spectrum of the polymer has a large negative peak at 278 nm, which is characteristic of the poly(dG)-poly(dG)-poly(dC) triplex. The polymer is resistant to DNase and interacts much more weakly with ethidium bromide as compared with the double-stranded DNA.
Kotlyar
Alexander
A
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978 Israel. s2shak@post.tau.ac.il
Borovok
Natalia
N
Molotsky
Tatiana
T
Klinov
Dmitry
D
Dwir
Benjamin
B
Kapon
Eli
E
D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004256:DNA Polymerase I / Q000378:metabolism*; D004996:Ethidium / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D018625:Microscopy, Atomic Force; D011089:Polydeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism
D011089:Polydeoxyribonucleotides; C051802:triplex DNA; D004247:DNA; D004256:DNA Polymerase I; D004996:Ethidium