![]() The protein–DNA complex was separated from the monomeric species by gel filtration and labeled with FlAsH as described in Materials and Methods. The hairpin DNA substrates were designed to resemble a DNA replication fork with a duplex region of 12 bp and a long, 28-base 3′ single-stranded extension. The protein was then hexamerized using either ssDNA (dT 30) or hairpin DNA substrates, which were prelabeled with Alexa 568 on their 5′ or 3′ end, in the presence of Mg 2+ and ADP, a condition that does not support substrate unwinding. For the FRET experiment, E1 was purified as a stable monomer. A higher FRET interaction occurs between “top”-labeled E1 (128–605) helicase and the 5′-labeled DNA than with the 3′-labeled DNA. ( G) Orientation of E1 (DBD+OD+HD, residues 128–605) helicase on a “replication fork” substrate. In this experiment, we used a shorter 3′-ssDNA tail (dT13) that resulted in a stronger FRET signal than using the longer 3′-ssDNA (dT28) tail. ( Right) High FRET interaction between “bottom”-labeled E1 (308–605) helicase and 3′-labeled DNA. ![]() ( F) ( Left) Low FRET interaction between “bottom”-labeled E1 (308–605) helicase and 5′-labeled DNA. ( Right) Low FRET interaction between “top”-labeled E1 (308–605) helicase and the 3′-labeled DNA. ( E) ( Left) High FRET interaction between the “top”-labeled E1 (OD+HD, residues 308–605) helicase and the 5′-labeled DNA. The labeling efficiencies of FlAsH on all three sites including the CCPGCC and CXXC were all above 95% (based on absorbance measurements using the extinction coefficient ε FlAsH,528 = 70,000 cm −1⋅M −1). Wild-type E1(128–605) without a FlAsH binding site (green curve) did not bind FlAsH after washes. ( D) Emission spectra were scanned using 2 μM of E1 helicases labeled with FlAsH via CCPGCC or CXXC. In addition, they are solvent-exposed and not part of a network of hydrogen bonds. These di-cysteine substitutions were introduced to the noncatalytic region, where the target residues in the helical region are spaced by two to three residues apart (4–6 Å) and show low sequence conservation. ( Right) Di-cysteine mutations (A483C and R486C) incorporated into the HD (“bottom” position, 308–605). ( C) ( Left) Di-cysteine mutations (A347C and A350C) introduced into the OD (“top” position, 128–605). ( B) Schematic diagram illustrating the location of the labels on the helicase. FlAsH binding sites (CCPGCC or CXXC) were introduced into the constructs as indicated. Orientation of E1 helicase on a “replication fork” substrate.
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