ring-shaped β 2 sliding clamp that confers processivity to pol III, and
the clamp loader complex (τ/γ) 3 δδ
0
ψχ [11]. The clamp loader is
not only responsible for loading the β 2 sliding clamp onto the DNA
but also mediates the interaction with pol III core and the DnaB
helicase via its τ subunits [12] and with SSB via its χ subunit [5–7,
13]. Whereas τ and γ are both encoded by the dnaX gene, the γ
variant does not contain the domains required for interaction with
the core and the helicase due to translational frameshifting
[11]. Therefore, up to three cores can be bound in the holoenzyme
depending on the number of τ proteins in the clamp loader complex [14]. Furthermore, the clamp loader interacts with the template/primer allowing ATP hydrolysis and release of β 2 onto the
primer of each Okazaki fragment. Since the clamp loader interacts
with the template strand only, both RNA and DNA primers can be
accepted [15].
The interaction between the χ subunit of the clamp loader and
SSB facilitates processive DNA synthesis under elevated salt conditions [5]. We previously found that SSB and SSB/ssDNA complexes have the same affinity for χ under these conditions [7]. In the
experiments of Glover and McHenry [13] using surface plasmon
resonance (SPR), the χψ subcomplex of the clamp loader was found
to interact with SSB with a similar affinity (K D ¼ 2.7 μM) as
determined by AUC for χ alone (K D ¼ 3.4 μM) [6], whereas the
affinity of the τ clamp loader for SSB in the presence of template/
primer was found to be 1000-fold higher (K D ¼ 3 nM). It has been
shown that in some cases results obtained by SPR differed significantly from those determined by other methods like isothermal
titration calorimetry, AUC, or fluorescence quenching [16]. Since
SPR requires the immobilization of one reaction partner to a
surface, effects may appear, like occlusion of binding sites, steric
hindrance of ligand binding, finite rate of mass transport of the
ligand, and, if charged matrices like carboxymethylated dextran are
used, nonspecific electrostatic adsorption. As AUC is a first principal method that avoids binding to surfaces, the interaction of τ
clamp loader with SSB-saturated template/primer seemed to be a
good model system to demonstrate how high-affinity interactions
can be investigated using analytical ultracentrifugation with fluorescence detection.
1.3 Specific
Fluorescence Labeling
For our experiments, we specifically labeled the clamp loader complex with a single fluorescein probe at the C-terminus of the ψ
subunit. As the clamp loader is involved in several interactions, e.g.,
with SSB, template/primer, DnaB helicase, and the core of pol III,
we decided to specifically label a part of the complex that is not
known to be involved in any interactions. In order to place a single
label, we modified our expression plasmid for the ψ subunit in such
a way that at the C-terminus of the resulting protein a Staphylococcus aureus sortase (SrtA) recognition site followed by maltose402
Andrea Bogutzki and Ute Curth
the clamp loader complex (τ/γ) 3 δδ
0
ψχ [11]. The clamp loader is
not only responsible for loading the β 2 sliding clamp onto the DNA
but also mediates the interaction with pol III core and the DnaB
helicase via its τ subunits [12] and with SSB via its χ subunit [5–7,
13]. Whereas τ and γ are both encoded by the dnaX gene, the γ
variant does not contain the domains required for interaction with
the core and the helicase due to translational frameshifting
[11]. Therefore, up to three cores can be bound in the holoenzyme
depending on the number of τ proteins in the clamp loader complex [14]. Furthermore, the clamp loader interacts with the template/primer allowing ATP hydrolysis and release of β 2 onto the
primer of each Okazaki fragment. Since the clamp loader interacts
with the template strand only, both RNA and DNA primers can be
accepted [15].
The interaction between the χ subunit of the clamp loader and
SSB facilitates processive DNA synthesis under elevated salt conditions [5]. We previously found that SSB and SSB/ssDNA complexes have the same affinity for χ under these conditions [7]. In the
experiments of Glover and McHenry [13] using surface plasmon
resonance (SPR), the χψ subcomplex of the clamp loader was found
to interact with SSB with a similar affinity (K D ¼ 2.7 μM) as
determined by AUC for χ alone (K D ¼ 3.4 μM) [6], whereas the
affinity of the τ clamp loader for SSB in the presence of template/
primer was found to be 1000-fold higher (K D ¼ 3 nM). It has been
shown that in some cases results obtained by SPR differed significantly from those determined by other methods like isothermal
titration calorimetry, AUC, or fluorescence quenching [16]. Since
SPR requires the immobilization of one reaction partner to a
surface, effects may appear, like occlusion of binding sites, steric
hindrance of ligand binding, finite rate of mass transport of the
ligand, and, if charged matrices like carboxymethylated dextran are
used, nonspecific electrostatic adsorption. As AUC is a first principal method that avoids binding to surfaces, the interaction of τ
clamp loader with SSB-saturated template/primer seemed to be a
good model system to demonstrate how high-affinity interactions
can be investigated using analytical ultracentrifugation with fluorescence detection.
1.3 Specific
Fluorescence Labeling
For our experiments, we specifically labeled the clamp loader complex with a single fluorescein probe at the C-terminus of the ψ
subunit. As the clamp loader is involved in several interactions, e.g.,
with SSB, template/primer, DnaB helicase, and the core of pol III,
we decided to specifically label a part of the complex that is not
known to be involved in any interactions. In order to place a single
label, we modified our expression plasmid for the ψ subunit in such
a way that at the C-terminus of the resulting protein a Staphylococcus aureus sortase (SrtA) recognition site followed by maltose402
Andrea Bogutzki and Ute Curth