Structure of RecA-DNA complexes studied by combination of linear dichroism and small-angle neutron scattering measurements on flow-oriented samples.
By combining anisotropy of small-angle neutron scattering (SANS) and optical anisotropy (linear dichroism, l.d.) on flow-oriented RecA-DNA complexes, the average DNA-base orientation has been determined in RecA complexes with double-stranded (ds) as well as single-stranded (ss) DNA. From the anisotropy of the two-dimensional SANS intensity representation, the second moment orientation function S is obtained. Knowledge of S is crucial for the interpretation of l.d. spectra in terms of orientation of the DNA bases and the aromatic amino acid residues. The DNA-base planes are essentially perpendicular to the fibre axis of the complex between RecA and dsDNA in the presence of cofactor ATP gamma S. A somewhat tilted base geometry is found for the RecA-ATP gamma S complexes with single-stranded poly(dT) and poly(d epsilon A). This behaviour contrasts the RecA-ssDNA complex formed without cofactor which displays a poor orientation of the bases. Well-ordered bases in the ssDNA-RecA complex is possibly reflecting the role of RecA in preparing a nucleotide strand for base-pairing in the search-for-homology process. While the central SANS intensity is essentially independent of the pitch of the helical complex, a secondary intensity maximum, which becomes focused upon flow orientation, is found to be a sensitive measure of the pitch. The pitch values for the complexes compare well with cryo-electron microscopy results but are slightly larger than those seen for uranyl-stained samples.
Nordén
B
B
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
Elvingson
C
C
Kubista
M
M
Sjöberg
B
B
Ryberg
H
H
Ryberg
M
M
Mortensen
K
K
Takahashi
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D016880:Anisotropy; D004247:DNA / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D009502:Neutrons; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases