From the Department of Human Biological Chemistry and Genetics and
the Sealy Center for Structural Biology, The University of Texas
Medical Branch at Galveston, Galveston, Texas 77555-1053
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INTRODUCTION |
The DnaB protein is the Escherichia coli primary
replicative helicase, i.e. the factor responsible for
unwinding the duplex DNA in front of the replication fork (1-3). The
enzyme is an essential replication protein in E. coli (4)
which is involved in both the initiation and elongation stages of DNA
replication (3, 5, 6). The DnaB helicase is the only helicase required to reconstitute DNA replication in vitro from the
chromosomal origin of replication. In the complex with
ssDNA,1 the DnaB protein
forms a "mobile replication promoter." This nucleoprotein complex
plays an activating role for the primase in the initial stages of the
priming reaction (4).
Sedimentation equilibrium, sedimentation velocity, and nucleotide
cofactor binding studies show that the DnaB helicase exists as a stable
hexamer in a large protein concentration range stabilized specifically
by magnesium cations (7-9). Hydrodynamic and EM data indicate that six
protomers aggregate with cyclic symmetry in which the protomer-protomer
contacts are limited to only two neighboring subunits (7, 10, 11).
Hydrodynamic and EM studies also provide direct evidence of the
presence of long range allosteric interactions in the hexamer,
encompassing all six subunits of the enzyme (7, 11, 12).
Recently, we obtained the first estimate of the stoichiometry of the
DnaB helicase-ssDNA complex and the mechanism of the binding (12, 13).
In the complex with ssDNA, the DnaB helicase binds the nucleic acid
with a stoichiometry of 20 ± 3 nucleotides per DnaB hexamer, and
this stoichiometry is independent of the type of nucleic acid base (9,
13). Our thermodynamic studies of binding of the DnaB hexamer to
different ssDNA oligomers show that the enzyme has a single, strong
binding site for ssDNA. Moreover, the same binding site is used in the
binding to oligomers, polymer DNA, and replication fork substrates (9,
12-14). Photo-cross-linking experiments indicate that the ssDNA
binding site is located predominately, if not completely, on a single
subunit of the hexamer (9, 12, 13).
Our data show that, in the complex with the replication fork DNA
substrates, the DnaB helicase preferentially binds to the 5' arm of the
fork (14). The 3' arm does not form a stable complex with the DnaB
hexamer associated with the 5' arm, and the 3' arm is in a conformation
in which it is accessible for the binding of another DnaB hexamer.
Moreover, the duplex part of the fork substrate does not significantly
contribute to the free energy of binding which predominantly comes from
interactions with the 5' arm (14).
Formulating a physical model of a hexameric helicase mechanism requires
the knowledge of the structure of the helicase-ssDNA complex. In phage
T7 helicase/primase and E. coli RuvB protein systems, EM
data indicated that, in the complex with the enzymes, the ssDNA passes
through the inner channel of the protein hexamer (15, 16). On the other
hand, an outside mode of ss nucleic acid binding has been proposed for
the hexamer of the SV40 T large antigen helicase (17).
In this communication, the structure of the DnaB helicase-ssDNA complex
has been studied using the fluorescence energy transfer method. We
present evidence that, in the complex with the DnaB hexamer, the ssDNA
oligomer, which occupies the entire total DNA binding site of the DnaB
helicase, passes through the inner channel of the hexamer. The results
indicate that in the stationary complex with the replication fork
substrate, the helicase does not invade the duplex part of the fork
beyond the first 2-3 base pairs.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Buffers--
All solutions were made with distilled
and deionized >18 megohms (Milli-Q Plus) water. All chemicals were
reagent grade. Buffer T2 is 50 mM Tris adjusted to pH 8.1 with HCl, 5 mM MgCl2, and 10% glycerol. The
temperatures and concentrations of NaCl and AMP-PNP in the buffer are
indicated in the text.
DnaB Protein--
The E. coli DnaB protein was
purified, as we described previously (18, 19). The concentration of the
protein was spectrophotometrically determined using the extinction
coefficient,
280 = 1.85 × 105
cm
1 M
1 (hexamer) (7).
Nucleic Acids--
All nucleic acids were purchased from Midland
Certified Reagents (Midland, TX). The 20 mer dT(pT)19,
labeled with fluorescein at the 5' end, or at a different location of
the nucleic acid, were synthesized using fluorescein phosphoramidate
(Glen Research). Labeling of 20 mers at the 3' end with fluorescein, or
labeling with rhodamine (Rh), was performed by synthesizing
dT(pT)19 with a nucleotide residue in a given location of
the nucleic acid with the amino group on a six-carbon linker and,
subsequently, modifying the amino group with fluorescein
5'-isothiocyanate or tetramethylrhodamine 6-isothiocyanate (Midland
Certified Reagents). The degree of labeling was determined by
absorbance at 494 nm for fluorescein (pH 9) using the extinction
coefficient,
494 = 7.6 × 104
M
1 cm
1, and at 555 nm for
rhodamine using the extinction coefficient,
555 = 8.0 × 104 M
1
cm
1 (9). Concentrations of all ssDNA oligomers have been
spectrophotometrically determined, using the nearest-neighbor analysis
(9, 20). The single-arm fork substrates were obtained by mixing the
proper oligomers at given concentrations, warming up the mixture for 5 min at 95 °C, and slowly cooling for a period of ~2 h (14).
Site-directed Mutagenesis of the DnaB Helicase--
Replacement
of the arginine residues at position 14 from the N terminus of the DnaB
protein and obtaining the DnaB protein variant, R14C, were performed
using the plasmid RLM1038, harboring the gene of the wild type DnaB
helicase.2 The site-directed
mutagenesis was accomplished in the NIEHS Center facility directed by
Dr. T. Wood.
Labeling the DnaB R14C Variant with Fluorescent
Markers--
Labeling of the six cysteine residues of the DnaB
variant, R14C hexamer, with CPM or fluorescein 5-maleimide was
performed in H buffer (50 mM Hepes/HCl, pH 8.1, 100 mM NaCl, 5 mM MgCl2, 10% glycerol)
at 4 °C. The fluorescent label was added from the stock solution to
the molar ratio of the dye/R14C
25. The mixture was incubated
for 4 h, with gentle mixing. After incubation, the protein was
precipitated with ammonium sulfate and dialyzed overnight against
buffer T2. Any remaining free dye was removed from the modified
R14C-CPM or R14C-Fl by applying the sample on a DEAE-cellulose column
and eluting with buffer T2 containing 500 mM NaCl. The degree of labeling,
, was determined by the absorbance of a marker using the extinction coefficient,
394 = 27 × 103 cm
1 M
1 for CPM
and
494 = 78 × 103 cm
1
M
1 for fluorescein, respectively. The
obtained values of
were 5.8 ± 0.1 for CPM and 5.7 ± 0.1 for fluorescein, indicating that all cysteine residues in R14C are
readily available for
modification.3
Fluorescence Measurements--
All steady-state fluorescence
titrations were performed using the SLM-AMINCO 48000S and 8100 spectrofluorometers (12, 21, 22). The emission spectra have been
corrected for instrument characteristics using the software provided by
the manufacturer. Fluorescence anisotropy measurements were performed
in the L format, using Glan-Thompson polarizers placed in the
excitation and emission channels. The fluorescence anisotropy of the
sample was calculated using (23)
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(Eq. 1)
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were I is the fluorescence intensity and the first
and the second subscripts refer to vertical (V) polarization
of the excitation and vertical (V) or horizontal
(H) polarization of the emitted light (19). The factor
G = IHV/IHH
corrects for the different sensitivity of the emission monochromator
for vertically and horizontally polarized light (24). The limiting fluorescence anisotropies of fluorophores, rlim,
were determined by measuring the anisotropy of a given sample at a
different solution viscosity, adjusted by sucrose or glycerol, and
extrapolating to viscosity =
, using the Perrin equation (19,
23, 25).
Determination of the Average Fluorescence Energy Transfer
Efficiency from Donors on the Small 12-kDa Domains of the DnaB Hexamer
to an Acceptor Located on the DNA Substrates--
The efficiency of
the fluorescence radiationless energy transfer, E, from
donors located on the small 12-kDa domains of the DnaB protein variant
R14C, to an acceptor located on a DNA substrate bound in the DNA
binding site of the DnaB helicase, has been determined using two
independent methods. The fluorescence of the donor in the presence of
the acceptor, FDA, is related to the fluorescence of
the same donor, FD, in the absence of the acceptor
by
|
(Eq. 2a)
|
where
D is the fraction of donors in the complex with
the acceptor, and ED is the average fluorescence energy transfer from a donor to an acceptor, determined from the quenching of the donor fluorescence. Thus, the average transfer efficiency, ED, obtained from the quenching of the
donor fluorescence is obtained by rearranging Equation 2a
|
(Eq. 2b)
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The values of
D have been determined using the
binding constants of a given DNA substrate for the DnaB helicase measured in the same solution conditions (9, 14).
In the second independent method, the average fluorescence transfer
efficiency, EA, has been determined, using a sensitized acceptor fluorescence, by measuring the fluorescence intensity of the acceptor (fluorescein or rhodamine), excited at a
wavelength where a donor predominantly absorbs, in the absence and
presence of the donor. The fluorescence intensities of the acceptor in
the absence, FA, and presence,
FAD, of the donor are defined as
|
(Eq. 3a)
|
and
|
(Eq. 3b)
|
where Io is the intensity of incident light,
CAT and CDT are the total
concentrations of acceptor and donor,
A is the fraction of
acceptors in the complex with donors,
A and
D are
the molar absorption coefficients of acceptor and donor at the
excitation wavelength, respectively,
FA and
BA are the quantum yields of
the free and bound acceptor, and EA is the average
transfer efficiency determined by the acceptor sensitized emission. All
quantities in Equations 3a and 3b can be experimentally determined. For
the case considered in this work, the acceptor is practically,
completely saturated with the donor, i.e.
A = 1. Thus, for
A = 1, dividing Equation 3b by 3a and rearranging
provides the average transfer efficiency as described by
|
(Eq. 3c)
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It should be pointed out that the energy transfer efficiencies,
ED and EA, are apparent
quantities. ED is a fraction of the photons absent
in the donor emission as a result of the presence of an acceptor,
including transfer to the acceptor and possible nondipolar quenching
processes induced by the presence of the acceptor, and
EA is a fraction of all photons absorbed by the
donor which were transferred to the acceptor. The true Förster
energy transfer efficiency, E, is a fraction of the photons
absorbed by the donor and transferred to the acceptor, in the absence
of any additional nondipolar quenching resulting from the presence of
the acceptor (23). The value of E is related to the apparent
quantities of ED and EA by
(26)
|
(Eq. 4)
|
Thus, measurements of the transfer efficiency, using both
methods, are not alternatives but parts of the entire analysis used to
obtain the true efficiency of the fluorescence energy transfer process,
E.
The fluorescence energy transfer efficiency between the donor and
acceptor dipoles is related to the distance, R, separating the dipoles by Equations 5a and 5b (23)
|
(Eq. 5a)
|
and
|
(Eq. 5b)
|
where Ro is the so-called Förster
critical distance (in angstroms), the distance at which the transfer
efficiency is 50%,
2 is the orientation factor,
d is the donor quantum yield in the absence of the acceptor,
and n is the refractive index of the medium
(n = 1.4) (23). The overlap integral, J,
characterizes the resonance between the donor and acceptor dipoles and
has been evaluated by integration of the mutual area of overlap between the donor emission spectrum, F(
), and the acceptor
absorption spectrum,
A(
), as defined by (23)
|
(Eq. 5c)
|
The fluorescence transfer efficiency determined for chemically
identical donor-acceptor pairs, characterized by the same donor quantum
yields, depends on the distance between the donor and the acceptor,
R, and factor
2, describing the mutual
orientation of the donor and acceptor dipoles (23). The factor
2 cannot be experimentally determined, however, the
upper,
max2, and the lower,
min2, limit of
2 can be estimated from the measured limiting
anisotropies of the donor and acceptor and the calculated axial
depolarization factors, using the procedure described by Dale et
al. (27). When both axial depolarization factors are positive,
max2 and
min2 can be calculated
from
|
(Eq. 6a)
|
and
|
(Eq. 6b)
|
where <dDx> and
<dAx>are the axial
depolarization factors for the donor and acceptor, respectively (27). The axial depolarization factors have been calculated as square roots
of the ratios of the limiting anisotropies of the donors and acceptors
and their corresponding fundamental anisotropies, ro
(ro = 0.4 for CPM and fluorescein) (19, 25, 27).
The parameter
2 can assume a value from 0 to 4. For
complete random orientation of the acceptor and donor,
2 = 0.67 (23). However, because the distance between a donor and an
acceptor depends upon the 1/6th power of
2, only the two
extreme values (0 or 4) would significantly affect the determined
distance. It should be pointed out that the analysis of the possible
range of distances between the donor and the acceptor, using
min2 and
max2, describes the situation
where only a single donor-acceptor pair is used. Another equally
rigorous procedure to evaluate the error in the distance determination,
although more time consuming and much more expensive, is to use
multiple donor-acceptor pairs (28). The different molecular structures
of the different donor and acceptors introduce intrinsic randomization
of the orientation of the absorption and emission dipoles. The
measurement of a very similar distance using a different donor-acceptor
pair, indicates that the orientation of the donor and absorption
dipoles is far from the extreme values of 0 or 4, and that the true
distance between a donor and an acceptor is very close to the distance obtained using
2 = 0.67.
In the system studied in this work, six fluorescent labels, arranged in
a ring, are located at one end of the DnaB hexamer (see below). A
fluorescence energy homo-transfer between the same fluorophore
molecules may occur in such a system which would complicate the
molecular distance estimates. The extent of the homo-transfer was
examined by determining the fluorescence anisotropy of the DnaB hexamer
as a function of the degree of labeling and by excitation fluorescence
anisotropy spectra of the fully labeled DnaB hexamer. None of these
approaches showed a measurable homo-transfer, even in the case of the
R14C-Fl labeled fluorescein which has a large overlap of its absorption
and fluorescence spectra (36).
Fluorescence Energy Transfer from Multiple Donors to a Single
Acceptor--
If a set of m identical donors transfers the energy to a
single acceptor, as in the cases studied in this work, the average transfer efficiency is weighted by the contributions,
Ei, from all donors and is defined in general as
|
(Eq. 7)
|
where Ei is the transfer efficiency from the
individual donor, i, to the acceptor. It should be noted
that, if all individual transfer efficiencies, Ei,
are equal, e.g. in the case where the donors are located at
the same distance from the acceptor, the experimentally determined
average fluorescence transfer efficiency is then E
(1/m)mEi = Ei.
Quantum Yield Determinations--
The quantum yields of
different chromophores used in this work,
, were determined by the
comparative method (29) we previously described (19). Quinine bisulfate
in 0.1 NH2SO4 and fluorescein in 0.1 NaOH were
used as a standard (absolute quantum yield
= 0.7 and 0.92, respectively) (30, 31).
 |
RESULTS |
Distance between the 5' End of the ssDNA and the Small 12-kDa
Domains of the DnaB Hexamer, Multiple Donor-Acceptor
Experiments--
The DnaB monomer, which has an elongated shape, is
built of two structural domains (7, 10, 32). A small 12-kDa domain at
the N terminus of the protein and a large 33-kDa domain at the C
terminus are both connected at the hinge region. This structure of a
monomer has been visualized in EM studies which also showed that, in
the cyclic DnaB hexamer, all protomers are oriented with the small
12-kDa domain in the same direction (10). In R14C-CPM or R14C-Fl, each
of the six small 12-kDa domains of the hexamer is labeled with a
fluorescent marker, coumarin (CPM) or fluorescein at a specific site
(see "Experimental Procedures"). Thus, all six fluorophores in the
labeled R14C variant are at the same end of the DnaB hexamer and
arranged in a ring. The schematic representation of the DnaB hexamer
based on hydrodynamic and EM data is shown in Fig.
1a.

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Fig. 1.
a, schematic representation of the DnaB
hexamer, based on hydrodynamic and EM studies (7, 10, 11). The hexamer
is built of six chemically identical subunits. Each of the DnaB
protomers has two structural domains connected at the hinge region, a
small 12-kDa domain at the N terminus of the protein and a large 33-kDa
domain at its C terminus. The protomers are oriented with the small
12-kDa domain in the same direction. In the DnaB variant, R14C,
arginine 14 at the N terminus is replaced by cysteine in the protein
providing a site for specific modification with the fluorescence donor
or acceptor on the 12-kDa domain of each of the DnaB protomers. The
ring of six fluorescent labels on the small 12-kDa domains in R14C-CPM
or R14C-Fl is depicted by the dark circles. b, oligomers of
ssDNA, dT(pT)19, labeled with fluorescein at a different
location on the 20 mer used in fluorescence energy transfer
experiments: 5'-Fl-dT(pT)19;
dT(pT)3-Fl-(pT)16;
dT(pT)8-Fl(pT)10;
dT(pT)15-Fl-(pT)4; and
dT(pT)19-Fl-3'. c, 5' single-arm replication
fork substrates, labeled at the 5' end of the arm and at the 3' end at
the duplex part of the fork, used in fluorescence energy transfer
experiments described in this work.
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Fluorescence energy transfer from a donor to an acceptor is one of the
most intensively used methods in studying macromolecular distances in
solution (23). The overlap of an absorption spectrum of an acceptor
with the emission spectrum of a donor is a condition for the
fluorescence resonance energy transfer to occur. The fluorescence emission spectrum of R14C-CPM (
ex = 425 nm) together
with the absorption spectrum of 5'-Fl-dT(pT)19 and
5'-Rh-dT(pT)19, as well as the fluorescence emission
spectrum of R14C-Fl (
ex = 485 nm), with the absorption
spectrum of 5'-Rh-dT(pT)19 in buffer T2 (pH 8.1, 20 °C),
containing 100 mM NaCl and 1 mM AMP-PNP, are
shown in Fig. 2, a-c. In the
case of all three donor-acceptor pairs, there is a very significant
spectral overlap of the donor emission with the acceptor absorption
spectrum, indicating that efficient fluorescence energy transfer can
occur, if the donor and acceptor are in close proximity.

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Fig. 2.
Spectral overlap between donor emission and
acceptor absorption spectrum in donor-acceptor pairs, studied in this
work, in buffer T2 (pH 8.1, 10 °C) containing 100 mM
NaCl, 1 mM AMP-PNP. a, R14C-CPM emission
spectrum (- - - - -) ( ex = 425 nm), absorption
spectrum of 5'-Fl-dT(pT)19 (------). b, R14C-CPM
emission spectrum (- - - - -) ( ex = 425 nm),
absorption spectrum of 5'-Rh-dT(pT)19 (------).
c, R14C-Fl emission spectrum (- - - - -)
( ex = 485 nm), absorption spectrum of
5'-Rh-dT(pT)19 (------).
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|
Fluorescence emission spectra of R14C-CPM (
ex = 425 nm),
in the absence and presence of unlabeled dT(pT)19, in
buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM AMP-PNP, are shown in Fig.
3. The presence of the unlabeled 20 mer
has very little effect on the CPM fluorescence intensity. The situation
is different in the case of the 5'-Fl-dT(pT)19 complex with
R14C-CPM. The emission spectra of the labeled nucleic acid in the
absence and presence of R14C-CPM, with the excitation at 485 nm, where
only fluorescein absorbs, are included in Fig. 3. The presence of
R14C-CPM causes an ~2 fold decrease of the fluorescence intensity of
5'-Fl-dT(pT)19, while saturation of the labeled 20 mer with
the unlabeled DnaB protein causes only an ~ 8% decrease of the
5'-Fl-dT(pT)19 fluorescence (spectrum not shown). Thus,
even in the absence of the energy transfer process, the presence of six
hydrophobic CPM residues affects the quantum yield of fluorescein
located at the 5' end of the ssDNA. The ratio of quantum yields of
5'-Fl-dT(pT)19, in the complex with R14C-CPM and free in
solution,
BA/
FA, has
been tested over a range of excitation wavelengths between 465 and 500 nm. In this spectral range of excitation, no detectable fluorescence
energy transfer from CPM residues to fluorescein occurs. The value of
BA/
FA is
constant and equals 0.51. This result is expected because the quantum
yield of fluorescein is independent of the excitation wavelength
between 400 and 500 nm (23). Thus, the ratio of quantum yields,
independent of excitation wavelength, reflects the change of the
emission intensity of 5'-Fl-dT(pT)19 resulting from the formation of the complex with R14C-CPM, in the absence of the energy
transfer process, and can be used to obtain the spectrum of
5'-Fl-dT(pT)19 in the presence of R14C-CPM, without the
changes induced by the energy transfer process, at any excitation
wavelength (see Equations 3b and 3c). Analogous effect of the presence
of the ring of six donor residues on the quantum yield of the acceptor at the 5' end of the 20 mer occurs in all studied complexes. The emission spectrum (
ex = 555 nm) of 5'
Rh-dT(pT)19, in the absence and presence of R14C-Fl, is
shown in Fig. 3. The quantum yield of Rhodamine is decreased by factor
0.65 in the complex with R14C-Fl, as compared with the free oligomer,
while the unlabeled DnaB protein causes only an ~9% decrease (data
not shown).

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Fig. 3.
Fluorescence emission spectrum of the DnaB
variant R14C-CPM in the absence (- - -) and presence
(-···-) of unlabeled dT(pT)19 ( ex = 425 nm) in buffer T2 (pH 8.1, 10 °C) containing 100 mM
NaCl and 1 mM AMP-PNP. Concentrations of R14C-CPM and
the oligomer are 9.6 × 10 7 M (hexamer) and
4.5 × 10 7 M (oligomer), respectively.
Fluorescence emission spectrum of 5'-Fl-dT(pT)19
( ex = 485 nm) in the absence (------) and presence
(- - - - -) of R14C-CPM (without energy transfer) in buffer T2 (pH
8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP. Concentrations of labeled dT(pT)19 and the protein
are 4.5 × 10 7 M (oligomer) and 9.6 × 10 7 M (hexamer), respectively.
Fluorescence emission spectrum of 5'-Rh-dT(pT)19
( ex = 555 nm) in the absence (- - -) and presence
(--- - ---) of R14C-Fl (without energy transfer) in buffer T2 (pH
8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP. Concentrations of 5'-Rh-dT(pT)19 and the protein
are 4.5 × 10 7 M (oligomer) and 9.6 × 10 7 M (hexamer), respectively.
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The sum of the independent emission spectra (
ex = 425 nm) of R14C-CPM and 5'-Fl-dT(pT)19 in the presence of an
unlabeled nucleic acid and R14C-CPM (without energy transfer),
respectively, in buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM AMP-PNP, is shown in Fig.
4a. The solid line in Fig.
4a is the fluorescence emission spectrum of the complex of
R14C-CPM with 5'-Fl-dT(pT)19 at the same concentrations of
the protein and nucleic acid as in the case of the sum of the
independent components of the complex. There is a dramatic difference
between the sum of the independent donor and acceptor spectra and the
spectrum where both donor and acceptor are placed in the same complex.
The emission intensity at the R14C-CPM maximum at 476 nm, in the
complex with 5'-Fl-dT(pT)19, is decreased by ~ 36%,
as compared with the R14C-CPM complexed with unlabeled
dT(pT)19. The decrease of emission at 476 nm indicates significant fluorescence energy transfer from the CPM residue located
on the small 12-kDa domain of the DnaB helicase to the fluorescein
moiety located at the 5' end of the bound
5'-Fl-dT(pT)19.

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Fig. 4.
a, sum of the fluorescence emission
spectra (- - -) of DnaB R14C-CPM in the presence of unlabeled
dT(pT)19 (4.5 × 10 7 M (oligomer)) and
5'-Fl-dT(pT)19 in the presence of R14C-CPM (without energy
transfer) ( ex = 425 nm) in buffer T2 (pH 8.1, 10 °C)
containing 100 mM NaCl and 1 mM AMP-PNP and the
fluorescence emission spectrum of the complex of R14C-CPM with
5'-Fl-dT(pT)19 ( ex = 425 nm) (------) in the
same buffer. Concentrations of 5'-Fl-dT(pT)19 and the
protein are 4.5 × 10 7 M (oligomer) and
9.6 × 10 7 M (hexamer), respectively.
The fluorescence emission spectrum of R14C-CPM normalized at 476 nm
(peak) to the emission spectrum of the protein in the complex with
5'-Fl-dT(pT)19 (--- - ---) is also included. Sensitized
emission spectrum of 5'-Fl-dT(pT)19 ( ex = 425 nm), in the complex with R14C-CPM (- -), obtained after
subtraction of the normalized spectrum of R14C-CPM superimposed on the
fluorescence emission spectrum of 5'-Fl-dT(pT)19 in the
presence of R14C-CPM (without energy transfer) obtained at the same
excitation wavelength by multiplying the spectrum of free, labeled 20 mer by the quantum yield ratio,
BA/ FA = 0.51. b, sum of the fluorescence emission spectra
(- - - -) of R14C-Fl in the presence of unlabeled
dT(pT)19 (4.5 × 10 7 M
(oligomer)) and 5'-Rh-dT(pT)19 in the presence of R14C-Fl
(without energy transfer) ( ex = 485 nm) in buffer T2 (pH
8.1, 10 °C) containing 100 mM NaCl and 1 mM
AMP-PNP and the fluorescence emission spectrum of the complex of
R14C-Fl with 5'-Rh-dT(pT)19 ( ex = 485 nm)
(------) in the same buffer. Concentrations of
5'-Rh-dT(pT)19 and the protein are 4.5 × 10 7 M (oligomer) and 9.6 × 10 7 M (hexamer), respectively. The
fluorescence emission spectrum of R14C-Fl normalized at 519 nm (peak)
to the emission spectrum of the protein in the complex with
5'-Rh-dT(pT)19 (- - - - -) is also included.
Sensitized emission spectrum of 5'-Rh-dT(pT)19
( ex = 485 nm), in the complex with R14C-Fl (- -),
obtained after subtraction of the normalized spectrum of R14C-Fl
superimposed on the fluorescence emission spectrum of
5'-Rh-dT(pT)19 in the presence of R14C-Fl (without energy
transfer) (- - - - -) obtained at the same excitation wavelength by
multiplying the spectrum of the free, labeled 20 mer by the quantum
yield ratio,
BA/ FA = 0.65.
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Comparison between the sum of the spectra of independent components of
the complex and the spectrum of the complex in Fig. 4a shows
that the fluorescence intensity of the fluorescein residue of
5'-Fl-dT(pT)19, with the peak at ~ 519 nm, is
strongly increased in the complex with R14C-CPM. Because fluorescein
does not contribute to the CPM emission band at 476 nm, we can
normalize the spectrum of R14C-CPM-unlabeled dT(pT)19 to
the spectrum of the R14C-CPM-5'-Fl-dT(pT)19 complex at 476 nm. The difference between the normalized spectrum of
R14C-CPM-unlabeled dT(pT)19 and the spectrum of the complex of R14C-CPM-5'-Fl-dT(pT)19 provides the sensitized emission
spectrum of 5'-Fl-dT(pT)19 saturated with R14C-CPM. The
emission spectrum of 5'-Fl-dT(pT)19 in the complex with
R14C-CPM (without energy transfer) and the sensitized emission spectrum
of 5'-Fl-dT(pT)19 in the complex with R14C-CPM, are
included in Fig. 4a. In the presence of the donor, CPM, the
fluorescence intensity of fluorescein at the 5' end of the bound 20 mer
is increased ~8.7-fold.
Experiments using a different donor-acceptor pair, fluorescein on the
12-kDa domain of the DnaB protein as the donor and rhodamine at the 5'
end of dT(pT)19 as the acceptor, are shown in Fig.
4b. The dashed line in Fig. 4b is the sum of the
independent emission spectra of the R14C-Fl and
5'-Rh-dT(pT)19 excited at
ex = 485 nm, where
fluorescein predominantly absorbs, in the presence of the unlabeled
nucleic acid and R14C-Fl (without energy transfer), respectively. The
fluorescence emission spectrum of the complex of R14C-Fl with
5'-Rh-dT(pT)19, at the same concentrations of the protein
and nucleic acid as the sum of independent components of the complex,
is shown as a solid line. Placing the donor and acceptor in the same
complex introduces a large difference in the donor and acceptor
emission spectra, as compared with the sum of the independent
components. In the complex with 5'-Rh-dT(pT)19, the
emission intensity of R14C-Fl, with the maximum at ~519 nm, is
decreased by ~23%, as compared with the protein spectrum complexed with unlabeled dT(pT)19. Also, the intensity of the
sensitized emission of rhodamine is ~23-fold higher than the emission
in the absence of the fluorescence energy transfer process. Both donor
quenching and strong sensitized emission indicate a significant fluorescence energy transfer from fluorescein on the small 12-kDa domain of the helicase to rhodamine located at the 5' end of the nucleic acid. The fluorescence energy transfer parameters for studied
donor-acceptor pairs are included in Table
I.
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Table I
Fluorescence energy transfer parameters for the R14C-CPM complex with
5'-Fl-dT(pT)19, the R14C-CPM complex with
5'-Rh-dT(pT)19, and the R14C-Fl complex with
5'-Rh-dT(pT)19, in buffer T2 (pH 8.1, 10 °C) containing 100 mM NaCl and 1 mM AMP-PNP
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The large effects on the observed spectral properties of the studied
complexes are reflected in the large values of the Förster energy
transfer efficiencies, E (Table I). In the case of the R14C-Fl complex with 5'-Rh-dT(pT)19, we obtained the
apparent transfer efficiencies of ED = 0.61 ± 0.04 and EA = 0.55 ± 0.04, respectively, using
Equations 2b and 3c. The difference between ED and
EA is within the experimental error of determination
of both quantities, nevertheless, it indicates that rhodamine, at the
5' end of the bound dT(pT)19, induces some additional
nondipolar quenching of the fluorescein emission. Larger differences
between ED and EA have been
obtained with other studied donor-acceptor pairs with the fluorophore
located at the 5' end of the 20 mer (Table I). The true Förster
fluorescence transfer efficiency from fluorescein, located on the small
12-kDa domains of the DnaB hexamer to the rhodamine residue at the 5' end of dT(pT)19, is then described by Equation 4 which
provides E = 0.59 ± 0.04. Analogous calculations
of the fluorescence energy transfer efficiency in the complex of
R14C-CPM with 5'-Fl-dT(pT)19 and 5'-Rh-dT(pT)19
provide E = 0.70 ± 0.04 and E = 0.55 ± 0.04, respectively (Table I).
The large values of the Förster energy transfer efficiencies for
all studied donor-acceptor pairs shows that the 5' end of the bound 20 mer, dT(pT)19, is in close proximity to the 12-kDa small
domains of the DnaB hexamer. In the first approximation, assuming
2 = 2/3 and using the determined value of
Ro(2/3) for the studied donor-acceptor pairs (Table
I), we obtained the average distances between the CPM residues on the
small 12-kDa domains of the DnaB hexamer and fluorescein at the 5' end
of the 20 mer R(2/3) = 45 Å. Analogous distances between
CPM residues and 5'-Rh-dT(pT)19 or R14C-Fl and
5'-Rh-dT(pT)19 are R(2/3) = 46 Å and
R(2/3) = 51 Å, respectively. The range of the possible
distance for each single donor-acceptor pair, determined using the Dale
et al. (27) analysis, is included in Table I. However, as we
pointed out, this uncertainty in the distance estimate would apply if
only a single donor-acceptor pair was used for the distance
determination. In the case where different donor-acceptor pairs are
used to determine the same distance, the different structures of the
fluorophores introduce additional randomization of the donor-acceptor
orientation not included in the Dale et al. analysis.
Determination of a similar distance in the macromolecular system, using
different donor-acceptor pairs, indicates the lack of a peculiar
orientation of the donor and acceptor dipoles which could significantly
affect the estimate of such distances through
2 to the
extent suggested by the Dale et al. method (see
"Experimental Procedures"). All studied donor-acceptor pairs in
this work provide a similar distance between the donor and the
acceptor, with the largest difference amounting to ~6 Å, with the
average value of 47 Å (Table I). Thus, the data indicate that the
obtained average distance between the 5' end of dT(pT)19,
bound to the single DNA binding site on one of the DnaB helicase
subunits, and the fluorophores, located at a specific site on the
12-kDa domains, is R = 47 ± 3 Å.
Fluorescence Energy Transfer from Donors on the 12-kDa Domains of
the DnaB Hexamer and the Acceptor Placed in a Different Location along
the ssDNA 20 Mer--
To obtain further insight about the topology of
the ssDNA-DnaB helicase complex, we performed fluorescence energy
transfer measurements of the distances between the ring of donors
located on the small 12-kDa domains of the DnaB hexamer and the
acceptor placed in different positions along the dT(pT)19
oligomer. The ssDNA oligomers used in these studies are depicted in
Fig. 1b. The fluorescence emission spectra
(
ex = 425 nm) of the complexes of R14C-CPM, with ssDNA
oligomers, dT(pT)19, each labeled with fluorescein at
different locations, at the 5' end, at the positions of 5, 10, 15, and
at the 3' end, are shown in Fig.
5a. Recall, fluorescein does
not contribute to CPM emission at 476 nm (Fig. 4a). To
facilitate a comparison, the spectra have been normalized to the same
degree of donor saturation with the protein (
D) and at 476 nm to represent a fraction of the free R14C-CPM emission at 476 nm. The
corresponding sensitized emission spectra of fluorescein, located at
different positions in the 20 mer, expressed as multiplicity of the
corresponding spectrum of the same free, labeled oligomer in the
complex with R14C-CPM (without energy transfer), are shown in Fig.
5b. The spectra in Fig. 5a show that quenching of
the CPM fluorescence, as a result of the presence of the acceptor on
the nucleic acid, occurs in each complex. The quenching of CPM
fluorescence is particularly significant in the complexes of R14C-CPM
with 5'-Fl-dT(pT)19 and
dT(pT)3-Fl-(pT)16 which differ in the location
of fluorescein (acceptor) by 5 nucleotide residues. Notice, the
quenching of CPM in the case of 5'-Fl-dT(pT)19 and dT(pT)3-Fl-(pT)16 is almost the same, despite
the fact that in dT(pT)3-Fl-(pT)16 the acceptor
is located five nucleotide residues further from the 5' end of the
nucleic acid. Dramatically diminished quenching is already observed in
the case of dT(pT)8-Fl-(pT)10, where the
acceptor is located by the distance of ten nucleotide residues further
from the 5' end of the nucleic acid, followed by complexes with
dT(pT)15-Fl-(pT)4 and
dT(pT)19-Fl-3' (Fig. 5a). The increase of the
intensity of the sensitized emission shows a very similar trend, with
the exception of dT(pT)3'-Fl-dT(pT)15 which has
a sensitized emission higher than 5'-Fl-dT(pT)19.

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Fig. 5.
a, fluorescence emission spectra
( ex = 425 nm) of the complexes of R14C-CPM with
dT(pT)19, labeled with fluorescein at different locations
along the ssDNA oligomer, in buffer T2 (pH 8.1, 10 °C) containing
100 mM NaCl and 1 mM AMP-PNP. The spectra have
been normalized to the same degree of donor (CPM) saturation with the
acceptor (fluorescein), D = 0.5: (------)
5'-Fl-dT(pT)19; (- - - - -);
dT(pT)3-Fl-(pT)16; (- - - -)
dT(pT)8-Fl-(pT)10; (--- ---)
dT(pT)15-Fl-(pT)4; (--- - ---)
dT(pT)9-Fl-3'. b, sensitized fluorescence
emission spectra of the fluorescein residue located at a different
position along the 20 mer, dT(pT)19, in the complex
with R14C-CPM ( ex = 425 nm). The spectra have been
normalized to the same degree of donor (CPM) saturation with the
acceptor (fluorescein), D = 0.5: (------)
5'-Fl-dT(pT)19; (- - - -)
dT(pT)3-Fl-(pT)16; (--- ··· ---)
dT(pT)8-Fl-(pT)10; (--- - ---)
dT(pT)15-Fl-(pT)4; (- - - - - -)
dT(pT)19-Fl-3'.
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The differences in spectral properties among the complexes are
reflected in the value of the fluorescence energy transfer efficiency,
E, which is only 0.11 ± 0.01 for the
dT(pT)19-Fl-3' and increases to 0.70 ± 0.04 for
5'-Fl-dT(pT)19 (Table II).
The corresponding average distance from the center of mass of the donors to the acceptors is 74 Å for dT(pT)19-Fl-3' and 45 Å for 5'-Fl-dT(pT)19 (see above).
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Table II
Fluorescence energy transfer parameters for R14C-CPM complexes with
dT(pT)19 oligomer labeled at different locations with
fluorescein (Fig. 1b), in buffer T2 (pH 8.1, 10 °C) containing
100 mM NaCl and 1 mM AMP-PNP ( ex = 425 m)
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It is interesting that the energy transfer efficiency,
E = 0.73 ± 0.04, observed in the case of
dT(pT)3-Fl-(pT)16, is even higher, although
within the experimental error range, than E = 0.70 ± 0.04 determined for 5'-Fl-dT(pT)15 (Table II). These
results indicate that fluorescein, located at the fifth position from the 5' end of the nucleic acid is located at the same, or even at a
shorter, distance from the plane of the donor ring as when it is placed
at the 5' end of the 20 mer. Recall, the donors, CPM residues located
at a specific site on each of the six 12-kDa domains of R14C-CPM, form
a ring whose plane is perpendicular to the longer axis of the hexamer
(Fig. 1a). The same, or shorter, distance from the donors to
fluorescein in dT(pT)3-Fl-(pT)16, as compared
with 5'-Fl-dT(pT)19, would result if the plane of the ring
of donors passes the nucleic acid axis around the third or fourth
nucleotide residue from the 5' end of the 20 mer. In such a structure,
the fluorescein attached at the 5' end of the nucleic acid would be
above the plane of the donor ring and separated from the plane of the
ring of donors by ~3-4 nucleotide residues, while fluorescein, in
the fifth position from the 5' end, would be below, or in, the plane of
the donor ring leading to the same or a higher energy transfer
efficiency. This conclusion is further supported by the fact that the
energy transfer efficiency from the donors and the next fluorescein
located at position 10 from the 5' end of the 20 mer is 0.22, a
dramatically lower value than 0.70 and 0.73 determined for
5'-Fl-dT(pT)19 and
dT(pT)3-Fl-(pT)16, although the distance
between the fifth and tenth position in the nucleic acid is very
similar to the distance between the 5' end and the fifth location in
the 20 mer (Fig. 1b, Table II) (see below).
Structure of the Complex of the DnaB Hexamer with ssDNA--
There
are two fundamentally different models which can describe the complex
between the DnaB hexamer, which has a single DNA binding site on one of
the protomers, and ssDNA. In the first model, the nucleic acid passes
through the inner channel of the protein hexamer. In this model, every
base of the bound ssDNA is, at the first approximation, at a similar
distance from each protomer of the hexamer. In the second model, the
nucleic acid binds to the single DNA binding site located on the
outside of one of the DnaB protomers. Thus, in this complex, there are
large differences among the distances between the nucleic acid and the protomers of the hexamer (see below).
EM studies show that the cyclic structure of the DnaB hexamer has a
diameter of ~140 Å, with the inner channel of the hexamer having a
diameter of ~40 Å (10). These dimensions indicate that the distance
from the center of the hexamer to the outside surface of the subunits
forming the hexamer is ~70 Å. On the other hand, the
Förster critical distances, Ro, for the used
donor-acceptor pairs are around 50 Å (Table I). In our fluorescence
energy transfer studies, the size of the DnaB hexamer and
Ro, of the used donor-acceptor pairs, impose the
first constraints on the possible distances between the donors and
acceptors and the values of the average energy transfer
efficiencies.
First, we consider the situation in which the acceptor on a nucleic
acid is located in the plane of the donor ring on the hexamer. In such
an arrangement, there is the shortest possible average distance between
the donors and the acceptor in both considered fundamental models of
the hexamer-ssDNA complex mentioned above. The simplified geometry of
both models, with the acceptor located in the center of the hexamer and
with the acceptor on the outside of one of the protein protomers, is
depicted in Figs. 6a and 6b. The cyclic
DnaB hexamer is approximated by the hexagon with the distance from the
outside surface to the center of the hexamer, b = 70 Å (10). The distance between the donors and the center of the hexamer is
designated as q. Because, in our experimental system, six
donors transfer energy to a single acceptor, the average energy
transfer efficiency, E, is described by Equation 7 (see "Experimental Procedures"). As we pointed out in the arrangement where the nucleic acid passes through the inner channel of the hexamer,
the acceptor, located on the nucleic acid, is at a similar distance,
R, from each donor in the donor ring. Therefore Equation 7
can be approximated by
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(Eq. 8)
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It should be pointed out, that with the acceptor located in the
same plane as the donor ring, R = q.

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Fig. 6.
a, schematic diagram showing the
arrangement of a ring of six donors, with each donor located at the
same site on a single protomer of a hexamer, and a single acceptor
located in the inner channel of the hexamer in the plane of the ring of
donors (the distance from the acceptor to the plane of the ring of
donors, x = 0). The distance, R, from the
acceptor to each donor is the same and equals the distance from each
donor to the center of the hexamer, i.e. R = q (see text for details). b, schematic diagram
showing the arrangement of a ring of six donors, with each donor
located at the same site on a single protomer of a hexamer, and a
single acceptor located on the outside of one of the protomers of the
hexamer, but in the plane of the donor ring (distance from the acceptor
to the plane of the ring of donors x = 0). In this
arrangement there are three different distances, R, from the
acceptor to each donor, as defined by Equations 10a-f. c,
geometrical relationships among the distance of donor to the center of
the hexamer, q, radius of the hexamer, b, and the
distances between the donors and the acceptor located on the outside of
one of protomers of the hexamer in the same plane as the ring of
donors, p and d (see text for details).
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For the model where the acceptor is located on the outside of one
of the protomers, the distances between the donors and the acceptor are
different, and the average energy transfer efficiency is defined
by Equation 7, which for this particular case is
|
(Eq. 9)
|
where R1, R2,
R3, R4,
R5, and R6 are distances
between a given donor and the acceptor defined as
|
(Eq. 10a)
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(Eq. 10b)
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(Eq. 10c)
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(Eq. 10d)
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(Eq. 10e)
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(Eq. 10f)
|
where p = (q/2)(30.5/sin
),
= 60
arctg{[(b
q)/(q + b)] 30.5}; d = (q/2)(30.5/sin
);
= 30
arctg{[(b
q)/(q + b)]3
0.5} (see Fig. 6c).
Notice, because the diameter of the DnaB hexamer is ~ 140 Å,
the distance from the donors to the center of the hexamer,
q, cannot be larger than ~70 Å. The theoretical
dependence of the average fluorescence energy transfer efficiency,
E, as a function of the distance between the donors and the
center of the hexamer, q, for both considered models of the
DnaB hexamer-ssDNA complex, are shown in Fig.
7, a and b. The
Ro selected in these simulations is 50 Å,
corresponding to the Ro of the studied donor-acceptor pairs (Table I). In the case where the acceptor is
located in the center of the hexamer, the average energy transfer efficiency increases as the distance between the donors and the center
of the hexamer decreases from E = 0.1 at
q = 70 Å and reaches the maximum possible value of
E = 1 at values of q below ~20 Å. Different behavior is observed in the case where the acceptor is
located on the outside surface of the hexamer. As the distance between
the donors and the center of the hexamer decreases, the average energy
transfer efficiency reaches a maximum of E = 0.22 at
q = ~40 Å, then decreases to E ~ 0.1, for donors approaching the center of the hexamer where the average
distance between the donors and the acceptors, q, is close
to 70 Å. The maximum value of E is dramatically lower, as
compared with the model where the acceptor is located in the center of
the hexamer (see above). It is evident that, for a cyclic hexamer, with
the radius of the hexamer ~70 Å, as in case of the DnaB hexamer,
and, with the critical Förster distance Ro = 50 Å, the average energy transfer efficiency cannot reach the value of
0.70-0.73, which is experimentally observed for the
DnaB-5'-Fl-dT(pT)19 and
DnaB-dT(pT)3-Fl-(pT)16 complexes, if the
acceptor is located on the outside of one of the hexamer subunits. Only
in the case where the radius of the cyclic hexagonal structure of the
hexamer is reduced to the physically unrealistic value of <40 Å,
corresponding to the diameter of the hexamer <80 Å, ~ two times
smaller than the value determined for the DnaB helicase, and with
q = 20 Å, i.e. all donors are located on
the inside surface of the inner channel of the hexamer, is the energy
transfer efficiency reaching the experimentally observed values of Å 0.7-0.75 (Fig. 7b).

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Fig. 7.
a, theoretical dependence of the average
fluorescence energy transfer efficiency from six identical donors,
arranged in a ring on the hexamer, to a single acceptor, placed in the
center of the hexamer in the same plane as the donor ring, upon the
distance between the donors and the center of the hexamer (------). The
donor-acceptor pairs have the same selected Ro = 50 Å; the maximum distance between the donors and the center of the
hexamer is 70 Å. Theoretical dependence of the average fluorescence
energy transfer efficiency from six identical donors, arranged in a
ring on the hexamer, to a single acceptor placed on the outside of one
of the protomers of the hexamer and in the same plane as the donor
ring, upon the distance between the donors and the center of the
hexamer (- - - -). The donor-acceptor pairs have the same
selected Ro = 50 Å; the maximum distance between
the donors and the center of the hexamer is 70 Å. b,
theoretical dependence of the average fluorescence energy transfer
efficiency from six identical donors, arranged in a ring on the
hexamer, to a single acceptor, placed on the outside of one of the
protomers of the hexamer and in the same plane as the donor ring, upon
the distance between the donors and the center of the hexamer, for
different diameters of the hexamer: (---) 70 Å; (--- - ---) 65 Å;
(- - - - -) 60 Å; (--- ··· ---) 55 Å; (- - - -) 50 Å; (·····) 45 Å; (- - - - -) 40 Å; (--- ---) 35 Å. The donor-acceptor pairs have the same selected
Ro = 50 Å. The solid perpendicular line
indicates the value of the average fluorescence energy transfer
efficiency that corresponds to the location of all donors on the inside
surface of the inner channel of the hexamer (q = 20 Å).
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Thus, the high values of the energy transfer efficiencies in the
DnaB-5'-Fl-dT(pT)19 and
DnaB-dT(pT)3-Fl-(pT)16 complexes, and the
analysis described above, provide the first strong indication that, in
the complex of the DnaB hexamer with ssDNA, the nucleic acid passes
through the inner channel of the protein hexamer.
We now extend our analysis to a more complex situation where a single
acceptor is located in any position along a nucleic acid lattice bound
to the hexamer. In other words, the acceptor location is not limited to
the plane of the donor ring, but can be placed above, in, and below
this plane. Such a situation corresponds to the fluorescence energy
transfer process in the complexes of R14C-CPM with dT(pT)19
labeled with fluorescein at different locations along the nucleic acid.
The approximate geometrical representations of the donor-acceptor
system, according to the model in which the ssDNA passes through the
inner channel of the hexamer, or is bound on the outside of the
protein, are illustrated in Fig. 8,
a-d. The subunits of the DnaB hexamer are represented by
six spheres forming a hexagon. Six donors, located at the same specific site on each subunit, are at the same distance, q, from the
center of the hexamer and arranged in a ring formed by the subunits. The nucleic acid is symbolized by a straight, thin ribbon passing through the inner channel of the hexagon or attached to the outside of
one of the protomers. In both models, the 5' end of the nucleic acid is
above the plane of the donor ring. The distance between the acceptor
placed in an arbitrary location on the ss nucleic acid, with respect to
the plane of the donor ring, is designated as x.

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Fig. 8.
a, schematic diagram showing the
arrangement of a ring of six donors, with each donor located at the
same site on a single protomer of a hexamer, and a single acceptor
located along a nucleic acid lattice bound in the inner channel of the
hexamer. The distance from the acceptor to the plane of the ring of
donors is designated as x. The distance, R, from
the acceptor to each donor is the same for all six donors (see text for
details). b, geometrical relationships between the distance
from the donors to the center of the hexamer, q, radius of
the hexamer, b, and the distances between the donors and the
acceptor located along a nucleic acid lattice bound in the inner
channel of the hexamer, R (see text for details).
c, schematic diagram showing the arrangement of a ring of
six donors, with each donor located at the same site on a single
protomer of a hexamer, and a single acceptor located along the ss
nucleic acid bound to the outside of one the protomers of the hexamer.
The acceptor can be placed above, in, or below the plane of the ring of
donors. The distance from the acceptor to the plane of the ring of
donors is designated as x. The distances from the acceptor
to a particular donor are R1,
R2, R3,
R4, R5, and
R6 (see text for details). d,
geometrical relationships between the distance from the donors to the
center of the hexamer, q, radius of the hexamer,
b, and the distances between the donors and the acceptor
located along the nucleic acid lattice bound on the outside to one of
the protomers of the hexamer p, d,
R1, R2, and
R3 (Equations 12a-f, see text for
details).
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As we pointed out, the dimensions of the DnaB hexamer and the
Förster critical distance of the used donor-acceptor pairs impose
the first constraints on the possible distances between the donors and
acceptors and the possible values of the energy transfer efficiencies.
The next constraint comes from the short length (20 nucleotides long)
of the ssDNA used in our experiments. Independent measurements of the
structure of the 20 mer in the complex with the DnaB helicase indicate
that the length of the bound dT(pT)19 is ~60 Å (data not
shown), thus, close to the length of one strand of dsDNA 20 base pairs
long in the B form (70 Å) (33). In the case where the ss nucleic acid
passes through the inner channel of the hexamer (Fig. 8, a
and b), the distance between each donor in the ring and the
acceptor, located in any position on the nucleic acid, is the same and
equals R, and the average energy transfer efficiency is
described by Equation 8. The theoretical dependence of the average
fluorescence energy transfer efficiency, E, as a function of
the average distance from the acceptor to the plane of the donor ring
is shown in Fig. 9a. In these
calculations, we selected a distance of 10 Å (~3-4 nucleotide
residues) for the length of the fragment of nucleic acid protruding
above the plane of the donor ring, similar to the distance indicated by our fluorescence energy transfer data (see above). The plots are partially superimposed as a result of the fact that for the same average distance of the acceptor to the donors, the transfer efficiency is the same for this geometry of the hexamer-ssDNA complex. However, for different values of q, the plots span different regions
of the average distance, R. Thus, for q = 70 Å the maximum value of the energy transfer efficiency is only ~0.2,
and R spans the region between 70 and 100 Å (
). As the
donors approach the center of the hexamer, the value of E
increases, reaching the value of 1 for q < 20 Å. The
analogous dependence of the average energy transfer efficiency,
E, upon the distance of the acceptor from the plane of the
donor circle, x, for different values of q, is shown in Fig. 9b. Recall, in this case, x spans
the distance of 70 Å which is the selected length of the ss nucleic
acid for all values of q in these simulations. However, the
transfer efficiency is higher as the distance between the donors and
the center of the hexamer decreases. The value of x = 0 corresponds to the point where the ssDNA crosses the plane of the donor
ring.

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Fig. 9.
a, theoretical dependence of the average
fluorescence energy transfer efficiency from a ring of six donors,
located at the same site on each of the protomers of a hexamer, to a
single acceptor located along the ss nucleic acid bound in the inner
channel of the hexamer, upon the average distance of the acceptor from
the plane of the ring of donors, R, for a different distance
of the donors from the center of the hexamer, q (Fig.
8a). The maximum distance from the outside surface of the
hexamer to its center, b, and the selected length of the nucleic acid,
x, are 70 Å. The plane of the ring of donors passes the
axis of the nucleic acid at a distance of 10 Å from its 5' end (Fig.
8a). The Förster distance for the donor-acceptor pair
is 50 Å: ( ) q = 70 Å; ( ) q = 50 Å; ( ) q = 35 Å; ( ) q = 20 Å;
( ) q = 10 Å. b, theoretical dependence
of the average fluorescence energy transfer efficiency from a ring of
six donors, located at the same site on each of the protomers of a
hexamer, to a single acceptor, located along the ss nucleic acid
lattice bound in the inner channel of the hexamer, upon the distance of
the acceptor from the plane of the ring of donors, x, for
different distances of the donors to the center of the hexamer,
q (Fig. 8a). All parameters and symbols are the
same as in Fig. 9a above.
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The schematic arrangement of the donors and the ssDNA, when the nucleic
acid binds on the outside of the one of the protomers, is presented in
Fig. 8, c and d. As in the previously considered model, the six donors are located at the same specific site on each
subunit of the helicase at the same distance, q, from the center of the hexamer. The nucleic acid, symbolized by a
straight, thin ribbon, is now tangent to the
outside surface of one of the helicase subunits. The geometry of this
model is more complex than the one previously considered. Inspection of
Fig. 8, c and d, shows that the distance between
the acceptor on the nucleic acid and the donors varies significantly
for each particular donor. For the model of the DnaB helicase-ssDNA
complex, in which the nucleic acid binds on the outside of the hexamer,
the average energy transfer efficiency is defined by Equation 7 and the
average distance between the acceptor and the donors is defined as
|
(Eq. 11)
|
However, R1, R2,
R3, R4,
R5, and R6, the distances
between a given donor and acceptor located on the ssDNA, are now
defined as
|
(Eq. 12a)
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(Eq. 12b)
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(Eq. 12c)
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(Eq. 12d)
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(Eq. 12e)
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(Eq. 12f)
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where p = (q/2)
(30.5/sin
),
= 60
arctg{[(b
q)/(q + b)]30.5}; d = (q/2)(30.5/sin
);
= 30
arctg{[(b - q)/(q + b)]3
0.5}. The geometrical relationships
between the donor-acceptor distances, R, and the dimensions
of the hexagon representing the DnaB hexamer are shown in Fig.
8d.
The theoretical dependence of the average fluorescence energy transfer
efficiency, E, upon the average distance of the acceptor from the donors, for a different distance of the donors from the center
of the hexamer, q, is shown in Fig.
10a, with b = 70 Å and Ro = 50 Å. Because of the complex
relationship between the Rav and the particular
donor-acceptor distance Ri, the plots are not
superimposed, contrary to the model where the ssDNA passes through the
inner channel of the hexamer. In other words, the same average distance
is not accompanied by the same average energy transfer efficiency.
Notice, that independent of the distance between the donors and the
center of the hexamer, the average energy transfer efficiency never
exceeds the value of ~0.25 (Fig. 10a). This dramatically
low value of E, as compared with the energy transfer
efficiency obtained for the model in which ssDNA passes through the
inner channel of the hexamer, results from the fact that the average
distance between the donors and the acceptor is always significantly
larger than the Ro (50 Å) of the donor-acceptor
pair for any value of q. The analogous dependence of the
average energy transfer efficiency, E, upon the distance of
the acceptor from the plane of the donor circle, x, for
different values of q, is shown in Fig. 10b.

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Fig. 10.
a, theoretical dependence of the
average fluorescence energy transfer efficiency from a ring of six
donors, located at the same site on each of the protomers of a hexamer,
to a single acceptor, located along the ss nucleic acid bound to the
outside of one of protomers, upon the average distance of the acceptor
from the plane of the ring of donors, R, for a different
distance of the donors from the center of the hexamer, q
(Fig. 8c). The distance from the outside surface of the
hexamer to its center and the selected length of the nucleic acid are
70 Å. The plane of the ring of donors passes the axis of the nucleic
acid at a distance of 10 Å from its 5' end (Fig. 8c). The
Förster distance for the donor-acceptor pair is 50 Å: (------)
q = 70 Å; (- - - - -) q = 50 Å;
(--- ··· ---) q = 35 Å; (- - - -)
q = 20 Å; (·····) q = 10 Å;
(--- - ---) q = 1 Å. b, theoretical
dependence of the average fluorescence energy transfer efficiency from
a ring of six donors, located at the same site on each of the protomers
of a hexamer, to a single acceptor, located along the ss nucleic acid
lattice bound to the outside of one of the protomers, upon the average
distance of the acceptor from the plane of the ring of donors,
x, for different distances of the donors to the center of
the hexamer, q (Fig. 8c). (------)
q = 70 Å; (- - - - -) q = 50 Å;
(---···---) q = 35 Å; (- - - - -)
q = 20 Å; (·····) q = 10 Å;
(--- - ---) q = 1 Å. All parameters are the same as
in a above.
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The computer simulations described above have been performed using
dimensions of the hexamer and the nucleic acid corresponding to the
dimensions of the DnaB hexamer and the ssDNA oligomer forming the
complex in our fluorescence energy transfer studies. Clearly, the two
fundamentally different models of the DnaB helicase-ssDNA complex
differ dramatically in the value of the average fluorescence energy
transfer efficiency for an arbitrary distance of the donors from the
center of the DnaB hexamer, q. Moreover, the size of these
differences well exceeds the errors due to approximations applied in
the analysis (see "Discussion").
The dependence of the experimentally determined fluorescence energy
transfer efficiency upon the corresponding average distance of
fluorescein, placed in different locations along the 20 mer, from the
CPM residues, located on the small 12-kDa domains of the DnaB helicase,
is shown in Fig. 11. The maximum of the
observed E is ~0.73, thus, it is much higher than
predicted by the model in which the ssDNA binds on the outside of the
hexamer for any physically realistic distance of the CPM residues from
the center of the DnaB hexamer. Only the model in which the nucleic
acid passes through the inner channel of the hexamer can describe the experimental data. Because we know the diameter of the DnaB hexamer (140 Å) and the length of the ssDNA in the complex with the protein (~60 Å), we can approximately estimate the distance of the donors (CPMs) from the center of the DnaB hexamer. The solid line
in Fig. 11 is the computer fit of the experimentally determined
fluorescence energy transfer efficiency, as a function of the
corresponding average distance of fluorescein from the ring of CPM
residues, using b = 70 Å, x = 60 Å,
and with the plane of the ring of CPMs passing the axis of the nucleic
acid at 10 Å (3-4 nucleotide residues) from the 5' end of the bound
20 mer. The model fits all experimental data points remarkably well,
with the value of q = 44 Å. Thus, the data indicate
that the CPM residues are at ~44 Å from the center of the inner
channel. Comparison with the diameter of the inner channel, ~40 Å (10), indicates that the CPM residues are located ~24 Å from the
surfaces of the DnaB protomers which form the inner channel of the
hexamer.

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Fig. 11.
The dependence of the average fluorescence
energy transfer from six CPM residues of R14C-CPM located on the small
12-kDa domains of the DnaB hexamer to the fluorescein placed at
different locations of the 20 mer dT(pT)19 (Fig.
1b). Solid line is the computer fit of the
average fluorescence energy transfer efficiency from a ring of six
donors, with each donor located at the same site on a single protomer
of a hexamer, to a single acceptor, located along the ssDNA 20 mer
bound in the inner channel of the hexamer, upon the average distance of
the acceptor from the plane of the donor ring. The distance from the
outside surface of the hexamer to its center b = 70 Å,
the length of the nucleic acid is 60 Å and the Förster distance
for the CPM-fluorescein donor-acceptor pair is Ro = 52 Å. The fitted distance from the donors to the center of the hexamer
is q = 44 Å. The plane of the ring of CPM residues
passes the axis of the nucleic acid at a distance of 10 Å from its 5'
end, as indicated by the fluorescence energy transfer measurements of
the R14C-CPM complexes with 5'-Fl-dT(pT)19 and
dT(pT)3-Fl-(pT)16.
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The obtained results strongly indicate that, in the complex with the
DnaB protein, the ssDNA passes through the inner channel of the protein
hexamer. Our fluorescence energy transfer data indicate, for the very
first time, the existence of such a hexameric helicase-ssDNA complex in
solution.
Complex of the DnaB Helicase with the 5' Arm of the Replication
Fork--
The DnaB helicase binds preferentially to the 5' arm of the
replication fork in a single orientation in which the small 12-kDa domains of the hexamer face the 5' end of the arm and the large 33-kDa
domains contain the entry site for the duplex part of the fork
(14).2 The fluorescence energy transfer studies of the
structure of the DnaB hexamer complex with the 5' arm of the
replication fork have been performed in an analogous way, as described
above for the DnaB-labeled 20 mer complexes. In these experiments, we
used R14C-CPM and the 5' single-arm fork substrate with fluorescein at
the 5' end of the arm or at the 3' end in the duplex part of the fork,
as depicted in Fig. 1c. The obtained values of the energy transfer efficiencies are included in Table
III.
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Table III
Fluorescence energy transfer parameters for the R14C-CPM complex with
the 5' single-arm fork labeled at the 5' end of the arm with
fluorescein (Fig. 1c) and for the R14C-CPM complex with the 5'
single-arm fork labeled at the 3' end of the duplex part of the fork,
(Fig. 1c) ( ex = 425 m)
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The Förster fluorescence transfer efficiency from CPM, located on
the small 12-kDa domain to the fluorescein residue at the 5' end of the
arm of the fork substrate, is E = 0.65 ± 0.04. This value of E is very similar to the one determined for
the complex with the 5' Fl-dT(pT)19 ssDNA oligomer (Table
II). Using Ro = 52 Å for this donor-acceptor pair,
the obtained distance from fluorescein at the 5' end of the arm of the
fork to the CPM residues on the 12-kDa domains is 47 Å. Thus,
independently of the presence of the duplex part of the fork, the
helicase forms the complex with the 5' arm in which the 5' end of the
arm is at a very similar distance from the small domains of the
hexamer, as in the complex with the 20 mer. In other words, the 5' arm
occupies the same binding site and in the same way as the ssDNA 20 mer
inside the inner channel of the helicase hexamer.
Quantitatively, very different behavior is observed in the case of the
complex of R14C-CPM with a 5' single-arm fork where fluorescein is
located at the opposite 3' end of the substrate on the same 30 mer
(Fig. 1c; Table III). The Förster energy transfer efficiency, E = 0.04 ± 0.01, is dramatically
diminished as compared with the complex where fluorescein is located at
the 5' end of the arm. Also, notice that this value of E is
significantly lower than E = 0.11 ± 0.01 determined for the complex with dT(pT)19-3'-Fl. The value
of E = 0.04 ± 0.01 indicates that the average
distance between the ring of donors on the small 12-kDa domains of the hexamer to the 3' end of the duplex part of the replication fork substrate is ~ 88 Å, as compared with 74 Å determined for the 3' end of the bound ssDNA 20 mer.
 |
DISCUSSION |
Elucidation of fundamental aspects of the structure of a hexameric
helicase complex with ssDNA in solution is of paramount importance for
understanding the mechanism of the enzyme activities. The hexamer of
the E. coli primary replicative helicase DnaB protein forms
a ringlike structure built of six chemically identical subunits (7).
Quantitative thermodynamic measurements show that, in the complex with
ssDNA, the DnaB hexamer occludes only 20 ± 3 nucleotide residues
(9, 12, 22). Moreover, photo-cross-linking studies indicate that
predominately, if not only, a single subunit of the hexamer is involved
in interactions with the nucleic acid in a stationary complex. These
studies show that the ssDNA does not wrap around the hexamer; however,
they are consistent with two fundamentally different modes of DNA
binding to the enzyme. In the first mode, the nucleic acid could bind
on the outside of one of the hexamer protomers. Such a mode of binding
has been proposed for the SV40 large tumor antigen, hexameric helicase (17). In the other mode, the DNA binds to one of the protomers while
crossing the inner channel of the cyclic hexamer. This mode of binding
was indicated for the phage T7 helicase/primase and E. coli
RuvB proteins (15, 16). The inner channel of the DnaB protein has a
diameter of ~40 Å which can accommodate a single ssDNA strand (10,
11).
A striking result of the fluorescence energy transfer measurements
described in this work is the very high fluorescence energy transfer
efficiency (E = 0.70-0.73) from the ring of six
donors, all located on the small 12-kDa domains of the hexamer, to an acceptor placed at the 5' end of the bound 20 mer, which encompasses the entire binding site of the enzyme. The value of E = 0.70 shows that 70% of the total absorbed energy by the donors is
transferred to the acceptor. This can be possible if each donor
transfers 70% of its absorbed energy to the acceptor, or if ~4.2 CPM
residues are completely quenched by the acceptor, while ~1.8 CPM
residues do not transfer their energy at all. The later option is
physically very unlikely, in light of the fact that the ssDNA is bound
to a single protomer and, moreover, each donor in the donor ring is
located on an independent protomer of the large cyclic hexamer whose
diameter is ~140 Å.
The shortest distance between the acceptor and donors in the donor ring
would occur if the acceptor is located in the plane of the donor ring
(Figs. 6 and 7). However, theoretical analysis of the energy transfer
process for two possible modes of ssDNA binding to the DnaB hexamer
shows that the fluorescence energy transfer efficiency from the donors
to the acceptor cannot exceed the value of ~0.25 (b = 70 Å, Ro = 50 Å) for the mode in which the ssDNA
is bound on the outside of one of the protomers. Thus, even in the case
where the acceptor is placed in the plane of the donor ring, the value
of the energy transfer efficiency is dramatically lower than the
experimentally observed 0.7-0.73. Only when the acceptor is placed in
the center of the hexamer is the energy transfer efficiency reaching
the experimentally observed values. These results clearly show that the
observed high energy transfer efficiency can occur only if the acceptor is at a similar distance from each of the donors in the donor ring,
i.e. when the ssDNA is bound in the inner channel of the hexamer.
Fluorescence energy transfer experiments, with the acceptor located
along the ss nucleic acid lattice, fully support the model of the
hexamer-ssDNA complex in which DNA passes through the inner channel of
the hexamer and provide additional information on the structure of the
complex. The energy transfer efficiency, in the case of
5'-Fl-dT(pT)19 (E = 0.7), is slightly
lower, as compared with dT(pT)3-Fl-(pT)16
(E = 0.73), where fluorescein is located five
nucleotide residues from the 5' end. Although this difference is still
within experimental error, we have systematically obtained a slightly
higher energy transfer efficiency for the complex with dT(pT)3-Fl-(pT)16, as compared with
5'-Fl-dT(pT)19. This very similar, or even higher,
E value indicates that the plane of the ring of donors
passes the nucleic acid axis around the 3rd-4th nucleotide from the 5'
end, resulting in a shorter distance and a higher E from the
ring of donors to fluorescein in
dT(pT)3-Fl-(pT)16 than to fluorescein at the 5'
end of the 20 mer. Thus, the results indicate that, in the complex, the
5' end of the bound ssDNA oligomer protrudes approximately 3-4
nucleotide residues (~10 Å) above the plane of the donor ring. On
the other hand, shifting the location of the acceptor by the same
distance of the next 5 nucleotide residues from the 5' end of the
nucleic acid results in a large ~3-fold drop of the energy transfer
efficiency to E = 0.22. Theoretical analysis of the
energy transfer process indicates that such a large drop is predicted
by the model in which ssDNA binds in the inner channel of the hexamer,
but not by the outside binding mode (Figs. 9 and 10).
The experiments, using different donor-acceptor pairs, show that the
distance from the acceptor located at the 5' end of the ssDNA bound in
the inner channel of the hexamer to the donor located on the 12-kDa
domain of each of the DnaB protomers is 47 ± 3 Å. Because the
experiments with 5'-Fl-dT(pT)19 and
dT(pT)3-Fl-(pT)16 indicate that the 5' end of
the ssDNA protrudes ~10 Å above the plane of the donor ring, simple
calculations (see Fig. 8a) show that each donor in the ring
is at an average distance of q
46 Å from the ssDNA
bound in the inner channel. This result is in excellent agreement with
the computer fit of the experimentally obtained dependence of the
average energy transfer efficiency upon the average distance between
the donor ring and the acceptor located along the nucleic acid which
provides q = 44 Å, using the known diameter of the
DnaB hexamer (140 Å) and the determined Förster critical
distance for the CPM-fluorescein system, Ro = 52 Å (Table I). The value of q = 44-46 Å shows that the
diameter of the donor ring is ~90 Å, thus, much larger than 40 Å,
the diameter of the inner channel of the hexamer, indicating that the
donors are located at a distance of ~25 Å from the surface of the
inner channel of the hexamer.
The distance from the donor ring to the acceptor at the 3' end of the
bound 20 mer is 74 Å (Fig. 11, Table II). Notice, this distance does
not include a fragment of ~ 10 Å of the nucleic acid and
acceptor (fluorescein) at the 5' end of the ssDNA which protrudes above
the plane of the donor ring. Knowing that the ssDNA is bound in the
inner channel of the hexamer, we can estimate (see Fig. 8a)
the distance from the plane of the donors to the acceptor at the 3' end
of the nucleic acid, i.e. the length of the nucleic acid
fragment, including the acceptor, which is 58 Å. Thus, the total
length of the bound 20 mer, including the fluorescein residues at the
5' and 3' ends, is ~68 Å. Because the length of the fluorescein
residues is comparable with the size of the nucleic base, which is
approximately 3 Å, the length of the 20 mer in the complex with the
DnaB helicase, without fluorescein residues, is ~62 Å. This estimate
is in agreement with the independent measurement of the length of the
20 mer, using the nucleic acid labeled simultaneously with a donor and
an acceptor.4 As we pointed,
out the length of 62 Å is only ~11% shorter than the corresponding
length of the 70 Å of one strand of dsDNA in the B form.
In our analysis of the fluorescence energy transfer data, we used the
diameter of the ringlike structure of the DnaB hexamer ~140 Å which
was determined in EM studies (10). It is still possible that, due to
some "flattening" of the protein hexamer in EM experiments, this
diameter is slightly overestimated. Thus, using our hydrodynamic data
we can estimate the lower limit of the DnaB hexamer diameter as ~111
Å (7, 13), which would correspond to the distance of ~56 Å from the
outside surface of the hexamer to its center. However, the analysis of
the energy transfer process using a diameter of 56 Å for the DnaB
hexamer clearly shows that the energy transfer efficiency cannot exceed the maximum value of ~ 0.38, for the outside binding mode of the ssDNA, even if the acceptor is assumed to be placed in the plane of the
donor ring and all donors are assumed to be on the inside surface of
the inner channel of the hexamer, i.e. q = 20 Å (Fig. 7b). It is evident that even in this most favorable
arrangement, which gives the highest possible energy transfer
efficiency for the outside binding mode, the value of E = 0.38 is much lower than the experimentally obtained 0.59 or 0.7-0.73
for the R14C-CPM complexes with the 20 mers labeled at the 5' end with
different fluorescent markers (Table I).
The acceptor, located at the 5' end of the 5' arm of the replication
fork substrate, is at a distance of ~47 Å from the donors located on
the small 12-kDa domains of the DnaB hexamer. This distance is, within
experimental accuracy, the same as the distance between the donors and
the 5' end of the bound 20 mer (Tables I and III). The results indicate
that both the isolated 20 mer, as well as the 5' arm of the replication
fork, occupy not only the same binding site, but are also bound in the
same way within the binding site which encompasses 20 ± 3 nucleotide residues (12). Thus, the 5' arm has a structure very similar
in length to the single strand of a dsDNA, when associated with the
helicase, and occupies most of the binding site. At this point, it
should be noticed that the fluorescence energy transfer efficiency in the case of the complex of R14C-CPM with the 5' single-arm fork, where
fluorescein is located at the 3' end in the duplex part of the fork, is
only 0.04 ± 0.01. This value is significantly lower than
0.11 ± 0.01 obtained for the 20 mer with fluorescein located at
its 3' end. Altogether, these results indicate that the enzyme, when
bound to the 5' arm of the fork, does not invade the duplex part of the
fork by more than 2-3 base pairs in its stationary complex,
i.e. without ATP hydrolysis. This conclusion is corroborated
by the fact that thermodynamic analysis of the helicase binding to
different fork substrates did not show any substantial contribution of
the duplex part of the fork to the free energy of binding of the
helicase (14).
Based on our results, a model of the hexameric DnaB helicase, in the
complex with a replication fork, is depicted in Fig. 12. The thermodynamic data show that
the helicase is preferentially bound to the 5' arm of the fork, while
the 3' arm is protruding in front of the enzyme (14). The DnaB is
oriented toward the duplex part of the fork with the large domains of
its six protomers, while the 5' end of the helicase is in close
proximity to the 5' end of the 5' arm. The enzyme is predominantly
bound to the arm of the fork with possibly only 2-3 base pairs of the
duplex part of the fork involved in the binding site. In the complex, the 5' arm of the fork passes through the inner channel of the hexamer.

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Fig. 12.
A cartoon representing our current model of
the DnaB hexamer bound in a stationary complex to the replication fork
based on the results obtained in our work. In the complex, the
helicase is preferentially bound to the 5' arm of the fork, while the
3' arm is protruding in front of the enzyme (14). The small 12-kDa
domains of the DnaB hexamer are in close proximity to the 5' end of the
5' arm, with the 5' arm of the fork passing through the inner channel
of the hexamer. The binding site which encompasses 20 ± 3 nucleotide residues is shown as a groove in the protomer engaged in the
interactions with DNA. For clarity, only contours of two protomers at
the front of the hexamer are shown. The helicase is oriented toward the
duplex part of the fork with the large domains of its six protomers. In
this stationary complex, the enzyme is predominantly bound to the arm
of the fork with possibly only 2-3 base pairs of the duplex part of
the fork involved in the binding site. The arrow indicates
the direction of translocation of the enzyme along the DNA lattice and
the unwinding reaction.
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The E. coli DnaB protein in solution forms a very stable
hexamer (7). In fact, this property distinguishes the DnaB helicase from other well studied hexameric helicases that exist in solution as a
mixture of different oligomeric forms and assemble into a hexamer when
bound to DNA, or in the complex with nucleotide cofactors (7, 34).
Thus, the simplest mechanism of ssDNA binding in the inner channel of a
hexameric helicase, which exists in solution as a mixture of different
oligomeric forms, would include assembling the enzyme into its
hexameric structure around the nucleic acid. In the case of a stable
hexamer, such as the DnaB protein, this simple model does not apply.
Moreover, contrary to a recent suggestion (35), our data clearly show
that the DnaB hexamer binds polymer, oligomer ssDNA, and DNA substrates
resembling the replication fork in the presence of the ATP
nonhydrolyzable analog AMP-PNP, i.e. without ATP hydrolysis
which could be used "to open" the hexamer (8, 9, 12-14).
Although the exact mechanism of the DnaB helicase binding to a ssDNA is
still unknown, the fact that the DNA is bound in the inner channel of
the DnaB hexamer suggests that this mechanism must include a
conformational change which leads to a transient opening of the
hexamer, in the absence of ATP hydrolysis. On the basis of what is
already known about the E. coli DnaB hexamer, the
following possibilities can be pointed out. Our quantitative studies of the oligomeric structure of the DnaB protein have shown that the stability of the hexamer is controlled by specific binding of
multiple magnesium cations (7). Therefore, it is possible that the
transient release of Mg2+ from one or two protomers, at
the initial stages of nucleic acid binding, induces local
destabilization of one of the subunit interfaces and allows the nucleic
acid to enter the inner channel. On the other hand, thermodynamic and
EM studies have shown that the DnaB hexamer undergoes dramatic global
conformational changes upon binding of the nucleotide cofactors (7,
11). These conformational changes, which involve large reorientations
of the hexamer subunits and are controlled by nucleotide binding and
release, could play a role in facilitating the entry of the ssDNA into
the inner channel of the hexamer. It is also possible that
coordinated binding and release of magnesium cations and
nucleotide cofactors may be simultaneously involved in the
"opening" of the DnaB hexamer. The mechanism of the nucleic acid
binding to the DnaB helicase hexamer is currently being examined in our
laboratory.
We thank Dr. T. Wood from the NIEHS Center
for his excellent work in obtaining the DnaB protein variant R14C, and
for our many discussions. We also thank Gloria Drennan Davis for her
help in preparing the manuscript.
This work is dedicated to Dr. Marian A. Bujalowski on the occasion of
his 75th birthday.