Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.
We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.
Clegg
R M
RM
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Federal Republic of Germany.
Murchie
A I
AI
Zechel
A
A
Carlberg
C
C
Diekmann
S
S
Lilley
D M
DM
D001483:Base Sequence; D002122:Calcium Chloride / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004735:Energy Transfer; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D005454:Fluorescence Polarization; D015636:Magnesium Chloride / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D012235:Rhodamines / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D005452:Fluoresceins; D012235:Rhodamines; D015636:Magnesium Chloride; D012965:Sodium Chloride; D004247:DNA; D002122:Calcium Chloride; D019793:Fluorescein