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- W3149014097 abstract "360-Pos Board B115 Studies of DNA Breathing and Helicase Mechanisms by Single Molecule (SM) Fret Between 6-MI and Cy3 in DNA Replication Fork Constructs Wonbae Lee1, John P. Gillies2, Huiying Ji3, Carey E. Phelps1, Davis Jose4, Peter H. von Hippel4, Andrew H. Marcus1. Oregon Center for Optics, Department of Chemistry and Institute of Molecular Biology, Univeristy of Oregon, Eugene, OR, USA, Department of Biochemistry, Univeristy of Oregon, Eugene, OR, USA, Department of Chemistry, Univeristy of Oregon, Eugene, OR, USA, Institute of Molecular Biology and Department of Chemistry, Univeristy of Oregon, Eugene, OR, USA. 6-methyl isoxanthopterin (6-MI) is a fluorescent nucleotide base analog of guanine (G) that can be site-specifically substituted for G at key positions within a DNA construct, with little or no disruption of the normal nucleic acid structure. DNA constructs fluorescently labeled with 6-MI have been useful in probing local base conformational changes in bulk solution experiments, but to date no experiments with 6-MI have been undertaken at the singlemolecule level.Major challenges towards achieving this goal include working with fluorophores with small extinction coefficients (e350 nm~ 12000M-1cm-1) in the UV, and the necessity to separate weak fluorescence from scattered laser excitation light. We here report sm experiments performed on 6-MI-labeledDNA constructs that were resonantly excited using the 351 nm line of an argon ion laser in combination with a home-built total internal reflection fluorescence (TIRF) microscope and split-screen CCD camera. To investigate the feasibility of such sm fluorescence experiments we used a long biotinylated DNA strand (91-mer) that provided binding sites for a short DNA molecule (23-mer) containing three 6-MI residues. From experiments on this substrate we were able to monitor three successive photo-bleaching steps, indicating the presence of the three 6-MI chromophores. We next developed a new Forster resonance energy transfer sm(FRET) system between 6-MI and the carbocyanine dye Cy3 site-specifically inserted into the DNA backbone of a fork construct. By performing experiments in bulk solution and at the sm level, wewere able to demonstrate smFRET between a 6-MI probe at the fork junction and a Cy3 backbone probewithin the dsDNAof the fork. We are now using this approach to study DNA unwinding mechanisms by the bacteriophage T4 helicase primase (primosome) complex." @default.
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- W3149014097 date "2014-01-01" @default.
- W3149014097 modified "2023-09-27" @default.
- W3149014097 title "Single Molecule Observation of Cyclization of Short DNA Duplex" @default.
- W3149014097 hasPublicationYear "2014" @default.
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