From the § Laboratory of Structural Biology, NIEHS,
National Institutes of Health, Research Triangle Park, North Carolina
27709, the Biomedical Engineering and Physical Sciences
Program, National Institutes of Health, Bethesda, Maryland 20892, and
the ¶ Department of Molecular Medicine, Institute of
Biotechnology, University of Texas Health Science Center,
San Antonio, Texas 78245
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ABSTRACT |
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The interaction between human DNA polymerase (pol
) and DNA ligase I, which appear to be responsible for the gap
filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N-terminal domain of
DNA ligase I and the N-terminal 8-kDa domain of pol
that interacts
with the DNA template and excises 5'-deoxyribose phosphate residue.
Intact pol
, a 39-kDa bi-domain enzyme, undergoes indefinite self-association, forming oligomers of many sizes. The binding sites
for self-association reside within the C-terminal 31-kDa domain. DNA
ligase I undergoes self-association to form a homotrimer. At
temperatures over 18 °C, three pol
monomers attached to the DNA
ligase I trimer, forming a stable heterohexamer. In contrast, at lower
temperatures (<18 °C), pol
and DNA ligase I formed a stable 1:1
binary complex only. In agreement with the domain mapping studies, the
8-kDa domain of pol
interacted with DNA ligase I, forming a stable
3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa
domain of pol
did not. Our results indicate that the association
between pol
and DNA ligase I involves both electrostatic binding
and an entropy-driven process. Electrostatic binding dominates the
interaction mediated by the 8-kDa domain of pol
, whereas the
entropy-driven aspect of interprotein binding appears to be contributed
by the 31-kDa domain.
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INTRODUCTION |
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The DNA repair pathway termed base excision repair (BER)1 purifies genomic DNA of damaged nucleotides and abasic sites arising from a variety of exogenous and endogenous sources (1). For example, altered bases arising from deamination and from alkylation by both endogenous (e.g. S-adenosylmethionine) and exogenous alkylating agents (e.g. methyl methane sulfonate and vinyl chloride) are repaired by BER (2-4). The abasic site is generated from these base lesions by either spontaneous or enzymatic cleavage of the N-glycosidic bond. In both prokaryotic and mammalian cells, the abasic site is repaired by a mechanistically similar BER pathway (5, 6). In this pathway, the abasic site is usually cleaved by a class II AP endonuclease, followed by the sequential actions of a DNA polymerase, a 2-deoxyribose-5-phosphate lyase (dRP lyase), and finally a DNA ligase (5, 7). BER can be distinguished from other DNA excision repair pathways by the relatively small repair patch produced in double-stranded DNA after incision at the abasic site and also by the fact that base lesions repaired by the BER pathway are generally limited to modifications that are less bulky than those lesions repaired by the nucleotide excision repair pathway.
In mammalian cells, there are at least two BER pathways, which have
been designated as follows: "short patch" or simple BER, in which
the repair patch is a single nucleotide; and "long patch" or
alternate BER, in which the repair patch is 2 to <13 nucleotides (5,
8). In the case of short patch BER, several lines of research have
recently confirmed a role for DNA polymerase (pol
) (5, 8-10).
The N-terminal 8-kDa domain of pol
functions as a dRP lyase
catalyzing a
-elimination reaction releasing dRP from the preincised
AP site in double-stranded DNA (7, 11, 12). This domain is also capable
of functioning as an AP site lyase catalyzing strand cleavage at intact
AP sites in double-stranded DNA (12). Polymerase
and DNA ligase I
have been found together in a BER-proficient complex isolated from
bovine testis (13). Since purified human pol
and DNA ligase I
interact in vitro (13), it seems likely that these enzymes
also bind to each other within the naturally occurring BER-proficient
complex. In addition, an interaction between pol
and AP
endonuclease, another component of the BER-proficient complex, has been
observed (14).
In this study, the interaction between pol and DNA ligase I has
been further investigated by two independent, complementary approaches.
Initially, the regions of these enzymes that are required for stable
complex formation were mapped by affinity chromatography. Subsequently,
equilibrium experiments were conducted in the analytical ultracentrifuge to characterize the molecular species formed as a
result of the stable interactions between pol
and DNA ligase I. Thermodynamic studies were also conducted in the analytical ultracentrifuge to gain further insight into the mechanism of binding.
In contrast to the studies with pol
and DNA ligase I, we could not
detect a stable complex between pol
and AP endonuclease in similar
experiments. Together, our results suggest intriguing new implications
as to how pol
and DNA ligase I function together to complete the
base excision repair pathway.
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EXPERIMENTAL PROCEDURES |
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Preparation of Affi-Gel Affinity Resins and in Vitro Binding Assay
Affi-Gel 10 beads (Bio-Rad) with either recombinant human DNA
ligase I or bovine serum albumin (BSA) as the ligand were prepared as
described (15). The affinity beads (20 µl of beads with 0.15 nmol of
either DNA ligase I or BSA as the ligand) were resuspended in 400 µl
of binding buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 20% glycerol) containing 2% dry milk and incubated
for 15 min at 4 °C. After the addition of 0.15 nmol of intact human
pol , the 8-kDa N-terminal domain, or the 31-kDa C-terminal domain
(16, 17), the incubation was continued for 1.5 h. Beads were
collected by centrifugation, washed with binding buffer, and then
resuspended in 15 µl of SDS-sample buffer. After heating at 70 °C
for 5 min, proteins were separated by denaturing gel electrophoresis
and transferred to a nitrocellulose membrane. After incubation with the
pol
antibody, antigen-antibody complexes were detected by enhanced
chemiluminescence (Pierce). Similar assays were carried out with
different versions of the 8-kDa domain containing the single amino acid
changes indicated in the figure legend.
Purification of GST and GST-pol
Human pol cDNA was subcloned into pGSTag (18). After
induction of plasmid-encoded GST and GST-pol
, cell lysates were prepared and protein expression was examined as described previously (19). Glutathione-Sepharose beads (Amersham Pharmacia Biotech) with
equal amounts of either GST or GST-pol
were produced (19).
Pull-down Assays with Glutathione-Sepharose Beads
DNA ligase I polypeptides were labeled in vitro by
coupled transcription and translation and partially purified by
ammonium sulfate precipitation (19). Labeled polypeptides (5 µl),
which were resuspended in buffer A (50 mM Hepes, pH 7.7, 100 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol, and 0.1% Nonidet P-40), were
incubated at room temperature for 30 min with glutathione-Sepharose 4B
beads (20 µl with either GST-pol or GST as the ligand) that had
been diluted to a final volume of 150 µl with buffer A. After the
beads were washed three times with 1 ml of buffer A, bound proteins
were separated by SDS-PAGE. Labeled proteins were detected in the dried
gel by autoradiography.
Protein Purification
Recombinant wild-type human pol and its 8-kDa and 31-kDa
domains were produced and purified as described (16, 17). AP endonuclease was purified as described previously (20). Human DNA
ligase I was purified from baculovirus-infected cells (21).
Analytical Ultracentrifugation
Analytical ultracentrifugation was performed in a Beckman XL-A analytical ultracentrifuge using either a 4-hole or 8-hole rotor at rotor speeds appropriate to the study. All experiments were begun at 2 or 3 °C and incremented by 3 or 4 °C (except as noted) following attainment of equilibrium until either 38 °C was reached or clear signs of protein degradation occurred. A total of six types of equilibrium runs were performed as described below.
Intact pol with AP Endonuclease--
An 8-hole rotor was
used at the speed of 15,000 rpm. The centrifuge cells were flushed with
argon prior to loading to prevent oxidation of the AP endonuclease by
the atmospheric oxygen in the unfilled portion of the channels. Two
concentrations of each AP endonuclease and pol
having
absorbencies ~ 0.2 and 0.3 at 280 nm were run, the remaining
three cells were loaded with mixtures of AP endonuclease and pol
in
molar concentration ratios of 1:1, 1:2 and 2:1. These mixtures had
absorbencies ~ 0.3 at 280 nm. Transmitted light intensity data
were collected over a range of temperatures between 2 and 18 °C in
steps of 4 °C.
Intact pol with DNA Ligase I--
Again, the 8-hole rotor
was employed but at 10,000 rpm. Two cells were loaded with two
concentrations of pol
having absorbencies ~ 0.22 and 0.34 at
280 nm; two other cells were loaded with DNA ligase I with
concentrations having absorbencies ~ 0.13 and 0.2 at 280 nm;
and the remaining cells were loaded with pol
-DNA ligase I mixtures
with molar concentration ratios of 1:1, 1:2, and 2:1, all having
absorbencies ~ 0.25. Transmitted light intensity data were again
collected over a temperature range of 2-34 °C in steps of 4 °C.
The data collected at 34 °C were discarded because protein
degradation became apparent.
The 8-kDa N-terminal Domain of pol --
A 4-hole rotor was
employed at 27,000 rpm, and the three cells were loaded with
concentrations having absorbencies of 0.15, 0.22, and 0.3, respectively, at 280 nm. Analysis of the data clearly showed that the
protein did not self-associate. Intensity scans were taken only at 2, 10, 18, 25, and 32 °C, and used for the experimental estimation of
the protein fragment partial specific volume. It is assumed that these
estimated values are more accurate than the compositional values since
they were determined experimentally in the centrifuge, with appropriate
buffers over the appropriate temperature range. They were subsequently
used in the analysis of the experimental data from the fourth
experimental study.
The 8-kDa N-terminal Domain of pol with DNA Ligase I--
A
4-hole rotor was employed at 12,000 rpm, and the three cells were
loaded with 1:1, 1:2, and 2:1 molar concentration ratios of DNA ligase
I and the 8-kDa domain. The absorbencies at 280 nm in all three cells
were between 0.2 and 0.3. Intensity scans were taken at temperatures
from 2 to 38 °C in steps of 3 °C. This proved to be the most
stable mixture as no protein degradation was observed below
38 °C.
The 31-kDa C-terminal Domain of pol --
A 4-hole rotor was
run at 17,000 rpm to equilibrium with three different protein
concentrations having absorbencies ~ 0.25, 0.35, and 0.45 at 280 nm, respectively. Intensity data were collected from 2 to 30 °C in
steps of 4 °C.
The 31-kDa C-terminal Domain of pol with DNA Ligase
I--
Three different protein concentration ratios were loaded and
run to equilibrium at 10,000 rpm. The molar concentration ratios were
1:1, 2:1, and 3:1 of 31-kDa domain to DNA ligase I with
absorbencies ~ 0.2, 0.3, and 0.4, respectively. Intensity data
were collected from 2 to 38 °C in steps of 4 °C.
Data Analysis
Transmitted light intensity data were collected in all cases, but both intensities and their corresponding derived absorbencies were used for the analysis. The reason for using both forms of the collected data lies in the recent demonstration that the customary analysis of absorbency data using least-squares estimation is not optimal due to the non-Gaussian character of the noise (22). The noise in the absorbency signal is the result of nonlinear transformation of the intensity signals, which are Gaussian. It has been shown that if intensity data were fitted directly, the estimation process would be optimal. A brief discussion of issues and mathematical methods are given in the Appendix, under "Direct Fitting of Transmitted Light Intensity in Analytical Ultracentrifugation." Although simulated data clearly demonstrated the superiority of fitting intensity data, this is the first time where the methodology has been employed for actual collected data. For these reasons, both approaches were employed at every analysis step and the results compared. Where association was found to take place, the corresponding equilibrium constants were estimated by least-squares fitting of appropriate mathematical models to both absorbency and transmitted light intensity data. The mathematical models represented assumed modes of association whose validity was tested by the quality of the fit; changes of Gibbs standard free energies were calculated from the equilibrium constant values and the changes in standard entropy, enthalpy, and heat capacity of association were determined from the temperature dependence of the values of the changes in free energy.
The mathematical models which best fit the collected data for each experiment, are given below. These models were used for the estimation of the relevant association parameters.
Intact pol --
The association model that emerged for the
intact pol
data was that of an indefinite isodesmic
self-association. The mathematical model for this type of association
is given in Equation 1.
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(Eq. 1) |
DNA Ligase I--
The association model that emerged from the
DNA ligase I (L) data was 3L L3. Such a monomer-trimer
system is mathematically modeled in Equation 2.
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(Eq. 2) |
DNA Ligase I with the 8-kDa Domain of pol --
In addition
to the monomeric forms of these proteins, the DNA ligase I trimer and
some form of a hetero-oligomer of the two proteins were observed. The
large size difference between the monomers and the fact that very small
amounts of monomers remained unbound made the problem mathematically
ill-conditioned. This necessitated the use of mass conservation
principles to mathematically eliminate one of the reference
concentrations. The mathematical form for this type of system may be
written as shown in Equation 3.
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(Eq. 3) |
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DNA Ligase I with Intact pol --
The system described under
"DNA Ligase I with the 8-kDa Domain of pol
" above had to be
modified slightly to allow for the indefinite self-association of the
pol
as described under "Intact pol
." Thus, the appropriate
mathematical model is shown by Equation 4.
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(Eq. 4) |
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The 31-kDa C-terminal Domain of pol --
Single species
analysis indicated the existence of species larger than the monomeric
form. Reasonable data fits could be obtained assuming monomer-dimer or
monomer-trimer self-associations but the best fits were obtained using
the isodesmic model of Equation 1. Consistent with the results for
intact pol
, the analysis was completed using Equation 1 as the
mathematical model.
The 31-kDa C-terminal Domain of pol with DNA Ligase
I--
The complete model given in Equation 4 was used for the
analysis with n = 1 or n = 3, only to
find out that the mathematical fits were consistently better when
KBLn approached zero indicating that no interprotein
heterogeneous interactions were taking place. Therefore, the analysis
was completed using Equation 4 with the heterogeneous term omitted.
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RESULTS |
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Identification of the Regions Required for Complex Formation
between DNA Ligase I and pol by Affinity
Chromatography--
Analysis of the structure of pol
, initially by
controlled proteolysis and more recently by x-ray crystallography, has
shown that this enzyme is composed of an 8-kDa N-terminal domain that binds to DNA template and carries dRP lyase activity and a 31-kDa C-terminal domain that contains the DNA polymerase active site. In this
study, using solution conditions similar to those previously employed
for the isolation of macromolecular complexes containing pol
and
DNA ligase I (13), we found that both intact pol
and the 8-kDa
N-terminal domain bind to Affi-Gel 10 beads with DNA ligase I as the
covalently attached ligand, but were not retained by beads with BSA as
the ligand (Fig. 1A). In
contrast, no significant binding of the 31-kDa domain to either
of the affinity beads was detected (Fig. 1A).
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Intact pol --
It is well known that pol
can
self-associate under conditions of lower ionic strength, but is a
homogeneous monomer at higher ionic strength, such as buffer with 150 mM KCl (24). Under the conditions used here, single species
analysis of the data revealed the existence of higher oligomers. To
resolve the actual species formed, we fit a variety of models
describing self-associations such as nB
Bn or
nB
2Bn
B2n for n = 2, 3, or 4 and where B is the protein monomer. For
some of the models, the quality of the fit was reasonable and the fit tended to improve with the complexity of the model. It was observed, however, that with increasing temperature, models that included higher
oligomers fit the data better. These observations led to the
interpretation that intact pol
undergoes indefinite
self-association whereby oligomers of all sizes are formed. In
particular, if the free energy of association is constant for the
addition of each additional monomer to the oligomeric chain, the
association is termed isodesmic. The corresponding mathematical model
(Equation 1) was fitted to the collected data. The fit was excellent at all temperatures with an association constant in the range 8,800 to
10,100 M
1. Fig.
2A shows a plot of Gibbs free
energy of association versus temperature, where it is seen
that the addition of each monomer to the oligomeric chain requires
between
4.9 and
5.4 kcal/mol. The plots in Fig. 2B
summarize the thermodynamic parameters of the association. There was a
significant temperature effect; changes in both entropy and enthalpy
decreased with temperature, and both are negative above 290 K. A
possible interpretation scenario may be proposed here, based on the
association strength variations with increased temperature; starting at
low temperatures, the association strength increases, which means that
higher oligomers will be forming at least up to about 20 °C.
Entropic changes are positive but decreasing with temperature. This is
interpreted as an indication of changes in protein hydration upon
self-association, since the association itself increases order and,
therefore, decreases entropy. Above 18-20 °C, however, the
association strength stays constant. The negative entropy change may be
attributed to another effect such as a different structural form of the
monomer, which could reflect, for example, a temperature-driven
conformational change. This scenario is consistent with the pol
association with DNA ligase I (see below). Also, the specific heat
capacity changes are negative and increase in magnitude with
temperature, indicating reduced capability for thermal interactions.
This in turn is an indication that association results in the burial of sites, consistent with a growing oligomer. The above results clearly imply the existence of two binding sites on the pol
molecule for
binding to itself. Symmetric positioning of two similar sites is
consistent with our results as the events of adding successive monomers
to the chain are energetically identical (Scheme
1B).
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The 8-kDa N-terminal Domain of pol --
The low molecular mass
of this protein dictated the relatively high speed (27,000 rpm) used in
this experiment. Excellent single species fitting of the data made it
clear that there was no self-association. This implies that the binding
sites for the self-association of intact pol
do not reside within
the N-terminal domain. Because of the non-associating behavior of the
8-kDa protein system, the collected data were used to fit for the
partial specific volume (
) of the protein. The
value thus obtained is expected to be more accurate
than the compositional value and was used in analyzing the
heterogeneous association of DNA ligase I with the 8-kDa domain. The
experimentally derived value for the buoyancy term (1
) was slightly higher than that calculated from the
compositional value, but its rate of increase with temperature was
somewhat smaller than the generally accepted empirical rate of
= 0.000425 cm3/g °C.
The 31-kDa C-terminal Domain of pol --
The mathematical
analysis of the collected data strongly pointed to an indefinite
isodesmic association as was the case for the intact molecule. This was
true at both low and high temperatures, as defined in the previous
section. An interesting observation was that the free energy of
association for the attachment of each monomer to the growing
oligomeric chain is
1 to
1.5 kcal/mol lower for the 31-kDa domain
than for the intact protein (data not shown). This could be interpreted
as some type of cooperativity in the presence of the 8-kDa domain.
Otherwise, the mathematical fits were consistently excellent at all
temperatures using the indefinite isodesmic model of Equation 1. The
result is consistent with the self-association binding sites residing
within the 31-kDa rather than the 8-kDa domain.
Human DNA Ligase I Self-association--
Little has been reported
about the self-associative properties of mammalian DNA ligase I. Previous studies on the hydrodynamic properties of mammalian DNA ligase
I revealed that, in buffers containing at least 150 mM
NaCl, it behaves as an asymmetric monomer with a molecular mass of 98 kDa (25), which is a single polypeptide enzyme of ~102 kDa molecular
mass. The present experiments indicate that human DNA ligase I
reversibly self-associates to form a stable trimer in a lower ionic
strength buffer (Scheme 1A). Excellent fits were achieved by
using the equilibrium model in Equation 2, both when fitting data from
individual cells and also for global fits of data from the two cells,
which were initially loaded with two different DNA ligase I
concentrations (Fig. 3). The association constant increased from approximately 6.7 × 109 to
15 × 109 M1 in the
temperature range examined. Fig. 3A is a plot of Gibbs free
energy of association for the DNA ligase I trimerization. It can be
seen from the plot that the free energy of association for the
attachment of each DNA ligase I monomer lies in the range between
4.03 kcal/mol at 0 °C and
4.85 kcal/mol at 38 °C. The excellent fit of the thermodynamic data is a reflection of the quality
of the raw experimental data, the fitting procedure, and the robustness
of the association. Fig. 3B shows the temperature variations
of changes in the standard thermodynamic parameters. The very large
T
S0 term combined with the
positive heat capacity change,
Cp, are consistent
with an association that is affected through electrostatic type forces
such as salt-bridge formations. The small positive entropy changes,
however, indicate some water displacement from the hydration layer upon
association. If the association is effected through salt-bridge
formation, the small enthalpic increases along with the small but
positive heat capacity changes are consistent with partially exposed
salt groups whose burial upon association keeps thermal interactions
weak.
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Mixture of DNA Ligase I and the 8-kDa Domain of pol --
Since
the 8-kDa domain of pol
does not self-associate, whereas DNA ligase
I forms a trimer, it was initially assumed that the DNA ligase I trimer
will persist in the presence of the 8-kDa domain which may or may not
attach itself to the DNA ligase I trimer. Therefore, a sequence of
models was employed to fit the experimental data. These models were of
the forms 3L + B
L3 + B, 3L + B
L3B,
and 3L + 3B
L3B3, where L represents DNA ligase I and B the 8-kDa domain. The quality of the fit favored the
latter model, but due to the small size difference between L (102 kDa)
and the BL complex (110 kDa) and the fact that the strong interaction
resulted in very small amounts of unbound monomers for at least one of
the loaded concentration ratios, the mathematical optimization problem
turned out to be ill-conditioned. This was reflected in the high
sensitivity of the optimization process to the initial parameter
estimates and to inconsistent reference concentration ratios at the
outcome. This problem was resolved by using the mass conservation
principle (26). Since the mass of the solute can be measured accurately
by integrating the zero-time absorbency scans, one or more of the
concentrations at the reference radii can be expressed in terms of the
remaining fitting parameters. Intensity scans are useful in this
instance since the radial locations of the cell menisci and bottoms can
be ascertained with greater accuracy than from absorbency scans. Here,
one of the reference concentrations was solved in terms of the
association constant and the other reference concentration. Thus, the
final equilibrium model that was used for parameter estimation is
mathematically expressed in Equation 3. The quality of fit was easily
superior for 3:3 heterohexamer formation, and the analysis was
completed using n = 3 at all temperatures. Values for
the equilibrium constants were used to compute the Gibbs free energy
change for heterohexamer formation as shown in Fig.
4A. It is seen that Gibbs free
energy varies between
32 kcal/mol and
35 kcal/mol as the
temperature increases to 32 °C. The energy for the attachment of
each 8-kDa monomer to the DNA ligase I trimer may then be computed
after the DNA ligase I trimerization energy is subtracted; the
resulting values vary from
6.65 kcal/mol to
6.85 kcal/mol with
increasing temperature. At 32 °C, this gives a Ka
105 M
1 for binding of each
8-kDa monomer to each DNA ligase I binding site (Scheme
2).
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Mixture of DNA Ligase I and Intact pol --
Species analysis
of the collected equilibrium data at lower temperatures failed to
indicate the presence of species larger than 300 kDa, which corresponds
to the DNA ligase I trimer. This suggested that interactions between
pol
and DNA ligase I, if they occurred, would probably be limited
to a one-to-one molecular species. Initially, the data were fit
assuming models that included no heterogeneous complex formation, but
allowed for the DNA ligase I trimer and various pol
oligomers. The
quality of fits was poor and deteriorated at higher temperatures where
it became clear that a species somewhat heavier than the DNA ligase I
trimer was present near the cell bottom. Similar results were obtained
with a model that assumed a single pol
molecule binding to the DNA ligase I trimer. When the 3:3 association model was included at higher
temperatures, all fits were excellent and all statistical measures of
the quality of fit were excellent. In contrast, below 18 °C the
association constants for 3:3 complex formation dropped to zero.
Further analysis revealed that the best fits were obtained when a 1:1
pol
- DNA ligase I complex was included in the model. The concern
that such species would not be distinguishable among the various pol
oligomers was alleviated by the fact that the size of the 1:1
complex lies between the (pol
)3 and (pol
)4 oligomers and the method is sensitive enough to
distinguish species whose molecular masses differ by more than 3 or
4%. With the 1:1 heterodimer as the model, all statistical measures of
the quality of fit improved and were excellent. The above results are
consistent with the interpretation that a stable heterohexamer forms at
higher temperatures (>18 °C) while at lower temperatures, a single
pol
molecule binds a single DNA ligase I molecule to form a stable heterodimer.
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Mixture of 31-kDa C-terminal Domain with DNA Ligase I--
Since
the binding site for the DNA ligase I lies on the 8-kDa domain of pol
, it should be expected that the remaining protein domain would not
have affinity for DNA ligase I. This was indeed the case. Consistently
excellent mathematical fits were obtained when no heterogeneous
associations were included in the mathematical model. This is
consistent with the complete absence of interprotein binding by the
31-kDa domain and with localization of DNA ligase I binding sites in
pol
, as already discussed above (data not shown).
AP Endonuclease and AP Endonuclease-pol Mixture--
After
equilibrium was established at a given temperature, the data were
analyzed while equilibrium was being attained at the next higher
temperature. Therefore, these experiments were discontinued at 18 °C
as it became apparent that AP endonuclease does not self-associate and
does not associate with pol
. Species analysis of the AP endonuclease and the AP endonuclease-pol
mixture data clearly showed that AP endonuclease remained a monomer while in the mixture and
that no species could be detected with a molecular mass of ~77 kDa or
its integer multiples, which would be the mass of a 1:1 or higher
complex (data not shown). These data are consistent with earlier
experiments that failed to detect an association between pol
and AP
endonuclease (13, 14).
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DISCUSSION |
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Repair of a uracil-containing DNA substrate by the "short
patch" or simple BER pathway can be catalyzed by a protein complex isolated from bovine testis nuclear extract (13). Two of the components
of this complex, pol and DNA ligase I, probably participate in the
repair of other base lesions, in particular alkylated bases and AP
sites. Interactions between pol
and AP endonuclease (14) and
between pol
and DNA ligase I (13) may underlie the stability and
activity of BER complexes. Using affinity chromatography, we mapped the
regions of pol
and DNA ligase I that are required for the stable
interaction of these enzymes. Interestingly, this interaction occurs
between the non-catalytic N-terminal domain of DNA ligase I and the
8-kDa domain of pol
that binds to gapped DNA and possesses dRP
lyase activity (7, 11, 12, 27).
A series of equilibrium experiments was conducted in the analytical
ultracentrifuge to further investigate associative behavior in solution
of human AP endonuclease, pol , and DNA ligase I. AP endonuclease
was found to neither self-associate nor associate with pol
. In
contrast, pol
was found to be a dynamic molecule that both
self-associates and associates with DNA ligase I. Intact pol
undergoes indefinite isodesmic self-association, forming oligomers of
many sizes; the 8-kDa N-terminal domain did not self-associate, whereas
the 31-kDa C-terminal domain self-associates indefinitely. We interpret
these data to mean that intact pol
possesses two binding sites that
can mediate pol
self-association, that these binding sites are
energetically identical, and that the sites are located in the 31-kDa
C-terminal domain of pol
, rather than in the 8-kDa N-terminal
domain (Scheme 1). These conclusions are consistent with a previous
study reporting the self-association of rat pol
at higher protein
concentrations (>10 µM) and low ionic strength (24).
DNA ligase I formed a stable trimer in the low ionic strength buffer
used in the equilibrium ultracentrifugation experiments. This could be
explained by the presence of two binding sites on the DNA ligase I
monomer for binding to itself. The fact that the trimer is a stable
complex and that no additional monomers attach to it suggests
non-symmetrical positioning of the binding sites on the molecule's
surface, as illustrated in Scheme 1. In agreement with the domain
mapping studies (Fig. 1), both intact pol and its 8-kDa domain
formed stable complexes with DNA ligase I in solution. The 31-kDa
domain of pol
appears to influence the interprotein association,
but itself does not possess a DNA ligase I binding site. Three 8-kDa
domain molecules associate with the DNA ligase I trimer, to form a
stable heterohexamer at all temperatures (Scheme 2). The association of
the 8-kDa domain with DNA ligase I was not accompanied by an increase
in entropy and is consistent with binding through electrostatic forces.
In support of this notion, changing any one of several basic amino acid
residues on the surface of the 8-kDa domain to a non-polar residue
inactivated DNA ligase I binding (Fig. 1B).
The interaction of the intact pol molecule with DNA ligase I
depended critically on the temperature of the solution. At lower
temperatures, we observed a 1:1 interprotein association only, while
above about 18 °C three pol
molecules bound to three DNA ligase
I molecules forming a stable heterohexamer (Scheme 3). This shift in
association mode for binding above 18 °C was accompanied by an
average increase in entropy, consistent with water dispersion upon
hydrophobic interactions. The implication of this result is that a
conformational change in the 31-kDa domain of pol
is
temperature-mediated. This conformational change allows DNA ligase I
trimerization to dominate the overall association mode of the system
and allows the 8-kDa domain in intact pol
to bind to the DNA ligase
I trimer. This is illustrated in Scheme 3. The proposed conformational
change in pol
has implications for regulating assembly of DNA
ligase I into the stable trimer. At lower temperature, pol
does not
bind stably to the DNA ligase I trimer. Instead, only the 1:1 pol
/DNA ligase I interprotein complex is formed.
To complete short patch BER, a single nucleotide is inserted, the 5'
dRP group is removed and the resultant nick is ligated (28). Since
these first two steps are catalyzed by pol and the final one by DNA
ligase I, it will be of interest to determine how the formation of a
stable complex, either the 1:1 or 3:3, heterodimer or heterohexameric,
respectively, between these enzymes influences these reactions. In this
regard, it is noteworthy that DNA ligase I, which initiates the
ligation reaction by transferring an AMP moiety from itself to the
phosphate at the 5' terminus, binds to the domain of pol
that
interacts with and processes the 5' terminus in the steps prior to
ligation.
In addition to participating in BER, DNA ligase I is the enzyme responsible for joining Okazaki fragments during DNA replication. In this process, DNA ligase I binds to proliferating cell nuclear antigen, an interaction that is also mediated by the non-catalytic N-terminal domain of DNA ligase I (15). Thus, the demonstration here that DNA ligase I forms a stable trimer in solution is particularly intriguing since proliferating cell nuclear antigen functions as a homotrimer that encircles DNA and tethers interacting proteins such as DNA polymerases and DNA ligase I to their DNA substrate. This raises the possibility that structurally similar multiprotein complexes may be involved in the gap-filling and ligation steps of lagging strand DNA replication and short patch BER. Further studies of these protein complexes and their interaction with DNA substrates should be informative.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM47251 and a grant from the San Antonio Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: LSB, NIEHS,
National Institutes of Health, 111 T. W. Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-3267; Fax: 919-541-2260; E-mail: wilson5{at}niehs.nih.gov.
The abbreviations used are:
BER, base excision
repair; pol , DNA polymerase
; dRP lyase, 2-deoxyribose-5-phosphate lyase; AP, apurinic/apyridimic; BSA, bovine
serum albumin; GST, glutathione S-transferase.
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APPENDIX |
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The measuring optical system in the analytical ultracentrifuge is the dual beam spectrophotometer which measures the transmitted light through the reference and the sample sectors of each loaded cell. Normally, the reference sector contains a buffer and the sample sector contains the solution, in the same buffer, of the macromolecules under investigation. The transmitted light intensity data of the reference sector, I0, and of the sample sector, Is, are customarily converted into light absorbances, A, using the simple relation, A = log10 (I0/Is). It is these transformed data that are fit to assumed concentration distribution models to obtain estimations of parameters such as molecular weights and association constants. This has been the established practice in the field due to the simple fact that absorbance is directly proportional to concentration whose distribution in the centrifugal field contains the information sought. Such apparently innocent transformation, however, has significance when viewed from the signal processing perspective. As with every measurement, the collected data are contaminated with random noise whose sources are rather diverse, including the noise in the electronic circuits, which contaminates both the light source and the sensor, the inaccuracies in the exact measurement position and, in all likelihood, the noise associated with the practical impossibility of obtaining absolutely clean quartz windows throughout both the reference and the sample window sectors. All these noise sources add stochastic noise to the data, and the noise from each source may even have different statistical characteristics. Some of these sources will add Gaussian noise, but other sources may result in different noise probability distributions. It is well known, however, from the central limit theorem (29) that, when a number of stochastic processes with differing statistical distributions are added together, the character of the resulting signal tends to be Gaussian or normally distributed. Therefore, it is expected that the raw collected data by the optical system in the centrifuge will be contaminated by Gaussian noise.
When scanning the cells in the XL-A ultracentrifuge, it is normal practice to scan each radial position a number of times and average the results. This reduces the noise in the recorded data. Again, the customary practice is to request the final recorded data in the form of absorbances to be analyzed later. The transformation of the intensity data into absorbances is a non-linear transformation and, as such, the noise characteristics are not preserved (29). At this point, the question of curve-fitting method for noisy data arises. The final decision for the most probable behavior of the solute in the particular solution is based on the quality of the fit as described by various statistical measures. The method used most often for curve fitting is that of least squares (30), which seeks estimates of the unknown model parameters so that the sum of squares of the deviations of the actual data from the model curve is minimized. In addition, it is known that optimal least-squares estimation is achieved if the data are weighted by the inverse of the variance. Hence, weighted least squares has been the method of choice. This, however, requires an iterative scheme as the optimal weights are not known at the outset. It is known from estimation theory (31) that the above method gives unbiased estimations of the unknown parameters under one of the following two conditions; either the noise probability distribution is Gaussian, or else no statistical information about the characteristics of the noise is available.
The claim about the nature of the noise in intensity and absorbance data was born out of actual data collected from the centrifuge and fitted by Gaussian distribution functions (32). It was found that the noise in the intensity data is indeed closely Gaussian with standard deviation that increases linearly with signal strength and that the absorbance data noise deviates in a statistically significant manner from being Gaussian.
Least-squares method or any other method chosen to perform
curve-fitting is effectively acting as a noise filter to eliminate measurement noise in order to extract the useful information contained in the signal. It has been proven that, if the noise probability distribution is known, the method referred to as the maximum likelihood estimation (33) results in the absolutely optimal de-noising of the
signal and in unbiased parameter estimation. In addition, if the noise
is Gaussian, the least-squares method and maximum likelihood are
exactly equivalent. For the case of absorbance data, however, it was
shown previously that the resulting probability distribution of the
non-linear transformation of Gaussian intensity data results in a new
distribution that is neither Gaussian and not even symmetric.
Therefore, using the least-squares estimation method would result in
statistically biased parameter estimators. The mathematical details
connected to the above discussion may be found in a previous
publication (32). It is, therefore, proposed that the intensity data be
used directly in a least-squares scheme. One of the methods proposed
there requires that the reference data be first smoothed using a cubic
spline function series, which can be optimally fit by the weighted
least-squares method. This will result in an algebraic description of
the reference sector data Io(r). The
sample side intensity data can subsequently be fit with the function
Is(r) = Io(r) 10C(r,), where
C(r;
) is the total concentration distribution
of the assumed interaction model for the macromolecules under
investigation; here, r is the radial position and
is the
vector of parameters whose values are to be estimated by the
fitting.
For the purpose of demonstrating the difference between fitting absorbance or intensity data, we performed simulations of a variety of systems and juxtaposed the results. In these simulations, a system was assumed and a mathematical model for the concentration distribution along the radius was written for a given set of system parameters such as molecular masses and association constants. Then, noise with characteristics similar to those observed in the XL-A was added to those mathematical models to simulate collected data in intensity form which were also converted into absorbances. Finally, the method of weighted least squares was applied with both the traditional absorbance data and with the intensity data to investigate how well the two approaches recover the initial system parameter values.
The following systems were investigated.
Solutions of Simple Monomers-- Molecular masses were recovered from simulated data with an error of the order of 3-5% from absorbance data, while the error with intensity data was consistently under 0.5%. This is significant, especially when the analysis is required to distinguish between species which are similar in size. Absorbance data would require at least a 10% difference in size, while intensity could distinguish sizes differing by as little as 1-2%.
Monomer-Dimer Association-- Here, the association constant was the main parameter of interest. The estimation error here depended on the strength of association. For the strongest association, the error when using absorbance data was of the order of 7-8%, and, for the weakest association, it was of the order of 5-6%. In contrast, the error when using the intensity data directly always stayed below 2-3%.
Two Different Proteins Forming a Heterodimer-- The association constants were recovered with an error of about 5% when using absorbances, while the error was reduced to 2-3% with intensity data fitting.
Thus, the preformed simulations clearly demonstrate the advantage of fitting transmitted light intensity data directly. There are additional advantages in using intensity data that were discovered in the process of the investigation. First, the smoothing of the reference data using the spline functions is virtually "cleaning" the reference sector window of particles that might adhere to the quartz window and cause unwanted signal fluctuations in that sector. Such fluctuations are often clearly seen in intensity data as local aberrations from the expected smooth sensitivity profile of the sensor photomultiplier cathode. The transformation into absorbances hides these fluctuations, which are often a source of a significant portion of the error. Another advantage is that with intensity data it is much easier to locate both the meniscus and the bottom of the cell in order to eliminate data that are contaminated by edge effects or by the region of non-linear sensitivity of the sensors. In the present investigation, both absorbance and intensity fitting were used throughout the study but the final results presented are those obtained using intensity. For the purposes of supporting the above arguments, Fig. A1 (top panel) shows an example of intensity data fitting of the pol
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Thermodynamic Analysis Methods
Thermodynamic analysis of the association can be performed based
on the relationship between the association constant
Kab for the interaction a + b
ab and the Gibbs free energy change,
G0(T).
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(Eq. 1A) |
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(Eq. 2A) |
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(Eq. 3A) |
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(Eq. 4A) |
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(Eq. 5A) |
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(Eq. 6A) |
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(Eq. 7A) |
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(Eq. 8A) |
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REFERENCES |
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