Structural Changes Measured by X-ray Scattering from Human Flap
Endonuclease-1 Complexed with Mg2+ and Flap DNA
Substrate*
Chang-Yub
Kim
,
Binghui
Shen
§,
Min S.
Park
, and
Glenn A.
Olah¶
From the
Life Sciences and ¶ Chemical Science
and Technology Divisions, Los Alamos National Laboratory,
Los Alamos, New Mexico 87545
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ABSTRACT |
Human flap endonuclease-1 (FEN-1) is a member of
the structure-specific endonuclease family and is essential in DNA
replication and repair. FEN-1 has specific endonuclease activity for
repairing nicked double-stranded DNA substrates that have the 5'-end of the nick expanded into a single-stranded tail, and it is involved in
processing Okazaki fragments during DNA replication. Magnesium is a
cofactor required for nuclease activity. We used small-angle x-ray
scattering to obtain global structural information pertinent to
nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1·34-mer DNA flap complex, and the D181A·34-mer DNA flap
complex. The D181A mutant, which has Asp-181 replaced by Ala,
selectively binds to the flap structure, but has lost its cleaving
activity. Asp-181 is thought to be involved in Mg2+
binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173-9176). Our data indicate that FEN-1 and the D181A mutant each
have a radius of gyration of ~26 Å, and the effect of
Mg2+ on the scattering from the proteins alone is
insignificant. The 34-mer DNA fragment was constructed such that it
readily forms a 5'-flap structure. The formation of the flap
conformation of the DNA substrate was evident by both the extrapolated
Io scattering and radius of gyration and was
supported by NMR spectrum and nuclease assays. In the absence of
magnesium, the FEN-1·34-mer DNA flap complex has an
Rg value of ~34 Å, whereas the D181A·34-mer
DNA flap complex self-associates, suggesting that a significant protein
conformational change occurs by addition of the flap DNA substrate and
that Asp-181 is crucial for proper binding of the protein to the DNA
substrate. A time course change in the scattering profiles arising from
magnesium activation of the FEN-1·34-mer DNA flap complex is
consistent with the protein completely releasing the DNA substrate
after cleavage.
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INTRODUCTION |
The 5'-flap structure is a common DNA structural intermediate
occurring during DNA replication, recombination, and repair (1). In
eukaryotic DNA replication, displacement of an upstream primer by an
incoming polymerase can result in the formation of a 5'-flap structure
(2, 3). The 5'-flap intermediates are also formed during
double-stranded break repair (4, 5), homologous recombination (6), and
excision repair (7-10). In DNA repair and replication activities,
structural recognition of the 5'-flap by specific DNA repair nucleases
is essential. The importance of the DNA metabolic reactions, involving
the structure-specific nucleases, is best illustrated by the human
genetic disorder xeroderma pigmentosum (11-13). This disease,
characterized by severe sensitivity to sunlight and a predisposition to
skin cancer, results directly from defects in the nucleotide excision
pathway. Mutation defects in the repair nucleases may be a point of
breakdown in this DNA repair pathway.
The design of a model flap DNA structure, similar to those conjectured
to occur in the nucleotide excision pathway, has led to the discovery
of human flap endonuclease-1
(FEN-1),1 which structurally
recognizes and cleaves the flap DNA structure (4, 14-17). FEN-1, an
~43-kDa Mg2+- or Mn2+-dependent
enzyme, demonstrates both 5'-flap structure-specific endonuclease
activity (1, 4, 7) and nick-specific 5'
3' exonuclease activity (4,
14, 17, 18). The exonuclease activity of FEN-1 is similar to the
function of the 5'
3' exonuclease domain of Escherichia
coli DNA polymerase I (16) and identical to the activity in RNA
primer removal that is necessary for in vitro mammalian DNA
replication. In its endonuclease role, FEN-1 recognizes the
phosphodiester backbone of a 5'-flap single strand and tracks down this
arm to the cleavage site, the junction where the two strands of duplex
DNA adjoin a single-stranded arm (1, 3). FEN-1 does not cleave bubble
substrates, single-stranded 3'-flaps, heterologous loops, or Holliday
junctions, but acting as an exonuclease, FEN-1 will hydrolyze
double-stranded DNA substrates containing a gap or 3'-overhang. FEN-1
endonuclease activity is independent of 5'-flap length, and
endonuclease and exonuclease activities cleave both DNA and RNA without
the need for accessory proteins (19). FEN-1 does, however, interact
with other proteins at the replication fork, including a DNA helicase
(20), the proliferating cell nuclear antigen (21-24), and possibly
replication protein A (RPA) (25).
The biological significance of the FEN-1 gene (RAD27 in
Saccharomyces cerevisiae and rad2 in
Schizosaccharomyces pombe) is emphasized by genetic analysis
in yeast. The yeast FEN-1 mutants display severely impaired phenotypes
such as UV sensitivity, deficient chromosome segregation, conditional
lethality, and accumulation in S phase (15, 26-29). The yeast
rad27 null mutant is a strong mutator, and the majority of
mutations found are duplications. This is probably because unexcised
flap strands in Okazaki fragments displaced by upstream DNA
polymerization are subsequently annealed to the downstream
complementary sequence. This part of the sequence will be duplicated in
the next generation of DNA replication (30). FEN-1 activity requires a
free 5'-end of the flap DNA strand. For instance, secondary
structure formation of the single-stranded DNA into a hairpin structure
is known to prevent the enzyme's function (31). At risk motif
sequences such as trinucleotide repeats have a higher probability to
form these structures. Indeed, the same FEN-1 null mutant displays
length-dependent CTG tract destabilization and a marked
increase in expansion frequency (32, 33). Thus, FEN-1 is a key enzyme
for maintaining genome integrity, and mutations in FEN-1 may give rise
to a number of genetic diseases such as myotonic dystrophy,
Huntington's disease, several ataxias, fragile X syndrome, and cancer
(30, 32).
As the role of FEN-1 in DNA replication and repair is becoming more
clear, it is important to structurally characterize this enzyme to
better understand how it functions either as an exo- or endonuclease.
To examine the structure-function relationship of FEN-1 in its nuclease
capacity, we have studied the effect of magnesium on its conformation
in aqueous solution, as observed by small-angle x-ray scattering
(SAXS). Experiments were also done with a D181A mutant of FEN-1, which
still selectively binds to the 5'-flap DNA structure, but has lost its
catalytic ability (34, 35). We show that no measurable structural
change was evident in either FEN-1 or the D181A mutant due to the
presence of Mg2+. A DNA fragment was constructed so that it
readily adopts a 5'-flap conformation. When measurements of FEN-1 and
the D181A mutant were performed in the presence of the 34-mer DNA flap
fragment, the FEN-1·34-mer DNA flap complex was seen to be more
compact than the D181A·34-mer DNA flap complex, which indicates that
the wild-type FEN-1·34-mer DNA flap complex is in a cleavage-ready conformation. A time course scattering measurement showed that magnesium was able to activate the cleavage of the FEN-1·34-mer DNA
flap complex, and the protein was found to completely release the
remaining single- and double-stranded portions of the DNA products.
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EXPERIMENTAL PROCEDURES |
Protein Purification and Sample Preparation--
Protein
expression and purification were essentially carried out according to
Nolan et al. (36). After FEN-1 was eluted from the column
using elution buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, and 300 mM histidine), it was further
dialyzed in Tris-HCl buffer, pH 7.9, containing 100 mM
NaCl, 10 mM 2-mercaptoethanol, and 10% glycerol and then
concentrated in a Centriprep-10 concentrator (Amicon, Inc.). Final
protein concentrations used were ~6 mg/ml in the SAXS study for both
the wild-type and D181A mutant proteins, in which 10 mM
Mg2+ was either present or absent. Wild-type and D181A
mutant protein concentrations were ~4.7 mg/ml for the protein·DNA
flap complex studies, and the protein and DNA had a 1:1 stoichiometric
ratio. Final concentrations were determined by the Bradford assay (46) using bovine serum albumin
-globulin as a standard. Binding of the
two proteins to the flap DNA substrate was confirmed by gel shift assay
after labeling the 5'-end of the substrate with 32P. The
purity of the concentrated protein samples was checked by
SDS-polyacrylamide gel electrophoresis and gave single bands as
illustrated in Fig. 1a. The flap endonuclease activity of
the proteins was assayed via a flow cytometry-based nuclease assay system (36) before and after each SAXS measurement. Activity was also
observed by time-resolved SAXS measurements by activating the
endonuclease with Mg2+ in the presence of the flap DNA substrate.
Preparation of the 34-mer DNA Flap Substrate--
An
oligonucleotide (5'-CCCCCCATGCTACGTTTTCGTATACGTTTTCGTA-3') was
synthesized by the solid-phase phosphoramidite method using an Applied
Biosystems synthesizer. The oligonucleotide was designed to form a
5'-flap substrate, which contains two Watson-Crick duplex arms folded
by TTTT loops (37) and a 10-base-long 5'-flap single strand (see Fig.
1b). It was purified by eluting the material through a POROS
R2/H reverse-phase chromatography column, followed by eluting through a
POROS HQ/M anion-exchange chromatography column if necessary, equipped
with a Bio-Cad Workstation 700E (PerSeptive Biosystems). The purity was
analyzed using a Tris borate/EDTA-urea gel (Novex) and found to be
>98% pure. NMR spectroscopic analysis was used to establish the
formation of the flap structure as shown in Fig. 1b.
Electrophoretic mobility shift and flap endonuclease assays were used
to determine the suitability of the newly designed substrate for
structural analysis.
SAXS Data Collection, Reduction, and Analysis--
SAXS data
were reduced as described elsewhere (38, 39) to give
I(Q) versus Q, where
I(Q) is the scattered x-ray intensity and
Q is the amplitude of the scattering vector. For elastic
scattering processes, Q is equal to 4
sin
/
, where
is half the scattering angle and
is the wavelength of the
incident and scattered x-rays. Guinier (40) and the indirect Fourier
transform (41) analyses were used to calculate Rg,
forward scatter (Io), and the vector distribution
function (P(r)). Aggregation was ascertained from
Io, which was expected to be proportional to the molecular mass (39). Lysozyme was used as a standard for scaling Io with the implicit assumption that lysozyme has
the same mean scattering density as FEN-1 and the D181A mutant.
Interparticle interference contribution to the scattering at the
concentrations used was assumed to be negligible since preliminary
measurements from the samples at concentrations between 0.5 and 6 mg/ml
scaled linearly and gave the same Rg values.
P(r) is the frequency of all interatomic vectors
within the scattering particle weighted by the product of their
scattering powers. The zeroth and second moments of
P(r) are equal to Io and
Rg, respectively, and the maximum linear dimension
of the scattering particle (dmax) was determined
from the corresponding value at which the P(r) function goes to zero.
SAXS measurements were done using the instrument described elsewhere
(38) at the Los Alamos National Laboratory. The instrument configuration used nickel-filtered 1.542 x-rays produced from a
1.5-kilowatt sealed tube copper target source and was line-focused by a
single mirror giving a full width at a half-maximum of 0.74 mm and a
full height of 26 mm as measured at the detector. A 4-inch-long position-sensitive linear detector (TEC Model 210Q) was placed 64 cm
from the sample. Measurements spanned a Q range of
0.015-0.27 Å
1. The net scattering from the samples was
calculated by subtracting a normalized buffer spectrum measured in the
same sample cell. Time course SAXS measurements, whereby data were
recorded for 30 min at subsequent intervals, was used to track
endonuclease activation after addition of magnesium to protein/DNA
substrate samples. Typically, measurements at these protein
concentrations require ~6 h for sufficient statistics; however,
30-min scans were good enough for observing changes in
Io. SAXS from at least two separate sample
preparations were measured for all measurements. Measurements were made
at 10 °C.
P(r) analysis of the data collected included a
deconvolution of the slit geometric contribution to the scattering.
Omission of this correction results in a systematic ~0.3-Å smaller
Rg for FEN-1, well within the statistics of the data
measured in this report (0.5-1.0 Å; 1 S.D.). Guinier analysis of the
data collected was not deconvoluted for instrument geometry.
 |
RESULTS AND DISCUSSION |
Magnesium is an essential cofactor for many enzymes involved in
DNA metabolism such as DNA polymerases, nucleases, and ligases. This
divalent metal, commonly ligated by acidic amino acid residues in
nucleases, attacks water molecules to produce a nucleophile that can
break phosphodiester bonds (34). Chelating of the metal ions out of
human FEN-1 can inactivate the enzyme completely. It has been
hypothesized that activation of the enzyme by adding Mg2+
to the reaction requires conformational changes in the enzyme before it
can cleave the DNA substrate (36). To test this hypothesis, we
performed small-angle x-ray scattering from wild-type FEN-1, the D181A
mutant, and their complex with DNA substrate to determine their global
structure and possible conformational changes upon addition of the
Mg2+ cofactor.
To perform SAXS experiments, it was necessary to develop an
experimental approach to produce a large quantity of flap DNA substrate. Unfortunately, the conventional approach, which utilizes annealing of three independent oligonucleotides, was inadequate for
this purpose due to its low yield. To overcome this problem, we
designed a single oligonucleotide that has a high propensity to form
the flap DNA structure, as shown in Fig.
1b (see "Experimental Procedures").

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Fig. 1.
Protein and DNA substrate
characteristics. a, purified wild-type FEN-1 and the
D181A mutant gave single bands when run on SDS-polyacrylamide gels.
b, the 34-mer DNA substrate was designed and synthesized
into an oligonucleotide that self-anneals into the 5'-flap, adjacent,
and template strands as shown.
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We used NMR spectroscopy and gel mobility shift and flap endonuclease
assays to determine that the newly designed oligonucleotide forms a
predicted flap DNA substrate. NMR spectroscopy showed the presence of a
double-stranded region registered by A=T and G
C base pairs and
showed the presence of a TTTT loop, and wild-type FEN-1 was able to
bind to DNA and yielded a correct cleavage product with an expected
size of the released flap strand (10 bases) (data not shown). Based
upon all of these results, we concluded that the new flap DNA substrate
could be used for our further study described.
Fig. 2 shows Guinier plots calculated for
FEN-1 and the D181A mutant as well as for FEN-1·34-mer DNA flap and
D181A·34-mer DNA flap complexes. Each sample gives a Guinier region
that can be fit with a straight line with reduced
2
below 1 (Table I). Molecular masses
calculated for the protein samples from Io and using
the lysozyme standard were within 10% of the expected value of 43,416 Da. In addition, there was no significant upturn at low Q in
the scattering profiles, except for the D181A·34-mer DNA flap
complex. The D181A·34-mer DNA flap complex sample had the same
concentration as the FEN-1·34-mer DNA flap complex and gave an
extrapolated Io value consistently ~10% larger
than that found for the FEN-1·34-mer DNA flap complex. This increase
in Io indicates slight aggregation of the D181A·34-mer DNA flap complex samples. The Rg and
dmax parameters in Table I were calculated from
combinations of two to four scattering measurements using different
sample preparations. Guinier plots of log(I·Q)
versus Q2 showed a linear
Q region (0.03-0.08 Å
1) with a negative
slope, from which the radius of gyration of cross-section
(Rc) could be calculated. A linear region in such a
plot suggests that the proteins have an elongated shape, at least in
one dimension. The Rc values are also tabulated in
Table I.

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Fig. 2.
Guinier plots of wild-type FEN-1 and D181A
mutant scattering data. Solid lines show the linear
fits defining the Guinier region. No upturns at low Q in the
scattering profiles, which could indicate aggregation, are seen except
for the D181A·34-mer DNA flap complex. This complex had the same
concentration as the FEN-1·34-mer DNA flap complex and gave an
extrapolated Io value consistently ~10% larger
than that found for the FEN-1·34-mer DNA flap complex, suggesting
that aggregation is present, but meager. This increase in
Io may be an indication of the importance of Asp-181
in proper binding of the enzyme to the DNA substrate. The plots were
offset for clarity of presentation and are labeled on the left along
the ordinate axis.
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Table I
X-ray scattering data from FEN-1 and D181A
Rg and Rc were calculated from
the scattering data using Guinier analysis. Rg and
dmax were calculated using
P(r) analysis. FEN-1 and the D181A mutant alone
give approximately the same Rg,
Rc, and dmax values in both the
absence and presence of Mg2+, suggesting that Mg2+
either induces no conformational change or induces a localized or
global conformational change that is not measurable within the
precision of these measurements. The 34-mer DNA in the presence of
Mg2+ aggregates as shown by a 16.6% increase in
Ip, therefore, the larger Rg and
dmax values observed relative to the case without
Mg2+ are not necessarily attributable only to a
Mg2+-induced conformational change in the DNA substrate. The
D181A · 34-mer DNA flap complex has a consistently ~10%
larger Io relative to the FEN-1 · 34-mer DNA
flap complex, suggesting that slight aggregation occurs in the
mutant · DNA substrate complex. Thus, the scattering data show
that the D181A mutant possibly binds and interacts differently with the
DNA fragment compared with wild-type FEN-1.
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Fig. 3 shows the Rg
values calculated from the P(r) analysis of the
individual measurements for the proteins alone in the presence and
absence of magnesium. Also, a comparison of the
P(r) functions is given in Fig.
4. The P(r)
functions have a single peak at ~27 Å and are fairly symmetric,
suggesting that the proteins are globular (ellipsoidal). Modeling the
data with one ellipsoid using a Monte Carlo modeling method described
elsewhere (42) gave dimensions for FEN-1 of a = 13.6 ± 0.2 Å, b = 32.4 ± 1.0 Å, and
c = 45.1 ± 1.0 Å for the best fit. The
scattering profile generated from this model also gave an
Rc value consistent with the Rc
value determined from the Guinier plots. Within the statistics of
Rg measurements, it is evident that magnesium has no
effect on FEN-1 or the D181A mutant in the absence of DNA. The fact
that Mg2+-induced conformational changes was not observed
is probably because the Mg2+ binding causes a localized
instead of global conformational effect or the induced global
conformational changes are quite small.

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Fig. 3.
Radius of gyration for wild-type FEN-1 and
the D181A mutant in the absence of the 34-mer DNA flap substrate and in
the presence or absence of Mg2+. The
Rg values are the same within the precision of these
measurements for both wild-type (WT) FEN-1 and the D181A
mutant and in the presence or absence of Mg2+. Conformation
changes induced by Mg2+ are not present or are localized or
global, but smaller than the precision of the SAXS measurements
described in this report.
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Fig. 4.
P(r) functions
for FEN-1 (a) and the D181A mutant (b) in the
absence of DNA. Black is in the absence of
Mg2+, and gray is in its presence. The
P(r) functions are fairly symmetric, indicating
that the two proteins are globular (ellipsoid shape) in solution and
are not affected by the presence of Mg2+ or by replacement
of Asp-181 by alanine. Single ellipsoid modeling against FEN-1 showed
it to have dimensions of a = 13.6 ± 0.2 Å,
b = 32.4 ± 1.0 Å, and c = 45.1 ± 1.0 Å.
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Next, we examine the effect of Mg2+ on the DNA substrate by
both scattering and modeling. Scattering profiles and
P(r) functions from the free 34-mer DNA fragment
in the absence and presence of Mg2+ are shown in Fig.
5. In the absence of Mg2+,
the P(r) function showed a peak at ~16 Å and
decreased approximately linearly out to ~60 Å. A simple two-cylinder
model was constructed based on the 5'-flap DNA structure shown in Fig.
1b. The intent of this modeling was to add further support
for the self-annealed structure by comparing the
P(r) function calculated from the model with the
P(r) function calculated from the scattering
data. In this model, one cylinder represents the double-stranded
portion of the DNA with dimensions of a B-type DNA structure. The
second cylinder represents the single-stranded portion and was assumed to have a diameter half that of a B-type DNA structure with a 4-Å
rise/base pair. One end of the second (smaller) cylinder was positioned
on the surface of the first cylinder and located at the middle of the
first cylinder along its axis so that the total DNA structure basically
forms a T shape. The cylinder axis of the second cylinder was then
given an angle of 45o relative to the cylinder axis of the
first cylinder. A schematic of the two-cylinder model is shown in Fig.
5b (inset). The P(r) function for this model is plotted in Fig. 5b and shows the
same basic features as the measurements, namely, a peak at ~17 Å and linearly decreasing long vectors. The scattering data are sensitive to
electron density pair distribution of vectors defining the scattering
object; however, phase information is lost in this type of measurement,
so a unique model cannot be determined based solely on a single
scattering curve. Therefore, the model having the same peak position at
~17 Å taken alone does not conclusively show that the 34-mer DNA
fragment adopts the 34-mer DNA flap, but it has not been ruled wrong.
On the other hand, this simple model, in combination with the binding
affinity measurements, the nuclease activity measurements, and the NMR
analysis, strongly supports that the 34-mer DNA fragment adopts the
flap structure. Addition of Mg2+ caused change in the
scattering profile and, more important, an increase by 16.6% in the
extrapolated Io scattering. This intensity increase
suggests that Mg2+ causes the DNA to aggregate. Aggregation
of the DNA substrate in the presence of Mg2+ is not
surprising since ionic interaction of the cation with the negatively
charged phosphate groups in the DNA backbone would decrease
electrostatic repulsion between DNA molecules in solution and allow
weaker attractive interactions to dominate.

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Fig. 5.
Scattering profiles (a) and
P(r) functions (b) for the free
34-mer DNA fragment in the absence (black circles) and
presence (gray squares) of Mg2+. The
P(r) function (b, dashed
line) calculated from a simple two-cylinder model can account for
the basic features seen in the P(r) function
measurement in the absence of Mg2+, supporting that the DNA
fragment adopts the flap structure. Addition of Mg2+ causes
the DNA fragments in solution to aggregate as evidenced by a 16.6%
increase in Io. This increase in
Io is evident in a. A schematic of the
two-cylinder model is shown in the inset of
b.
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A comparison of the scattering from the two proteins bound to the
34-mer DNA substrate is shown in Fig. 6.
Lysozyme could not be used as a standard for molecular mass
determination for the protein·DNA complexes as done for the proteins
alone since the mean scattering density for DNA is different from that
for protein. Nevertheless, we can still estimate the expected
Io for the protein·DNA complexes by comparison
with Io for the protein alone samples. We can write
the zero-angle scattering for the protein·DNA complex in terms of the
two components as follows (Equation 1),
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(Eq. 1)
|
where n is the particle number density, 
x
is the scattering density of component x, and
dVx is the integration volume element over component
x. Assuming specific volumes of 0.73 ml/g for the protein
and 0.56 ml/g for the nucleic acid, we expect, for our measurements,
that the contribution of the DNA would increase Io
by ~60% above the protein-alone measurement. This calculated
increase in Io takes into account the concentration
differences of the samples (~4.7 instead of ~6 mg/ml). We observed
this increase for the FEN-1·34-mer DNA flap complex samples,
suggesting that these samples are monodisperse. In addition, as
expected for monodisperse solutions, we obtained good agreement between
the Rg values obtained using the Guinier and
P(r) analyses, 34.4 Å (see Table I). The major
peak in the P(r) function for the wild-type
complex is ~28 Å, and dmax is ~104 Å.
However, a difference was observed between the Rg values for the D181A·34-mer DNA flap complex samples obtained from
the Guinier and P(r) analyses, 40.6 and 43.9 Å,
respectively, suggesting that the D181A·34-mer DNA flap complex
samples may be aggregated. The Io value is more
meaningful in evaluating the degree of aggregation of the
D181A·34-mer DNA flap complex and is ~10% above the
Io value for the FEN-1·34-mer DNA flap complex
samples, suggesting that aggregation is present, but not severe. In
addition, the dmax value calculated for the mutant complex samples is ~34 Å longer than that calculated for the
wild-type complex. It is possible that the D181A mutant complex is a
slightly more extended structure; however, drawing this conclusion is
precarious due to the slight aggregation problem. The scattering data
from the complexes and from the free proteins taken together suggest
that the D181A mutant binds and interacts differently with the DNA
fragment compared with wild-type FEN-1 and is evident by the induction
of aggregation of the mutant complex.

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Fig. 6.
Scattering profiles (a) and
P(r) functions (b) for FEN-1 and
the D181A mutant bound to the 34-mer DNA flap substrate in the absence
of Mg2+. Black circles indicate the
FEN-1·34-mer DNA flap complex, and gray squares indicate
the D181A·34-mer DNA flap complex. The FEN-1·34-mer DNA flap
complex samples were found to be monodisperse in solutions; however,
the D181A·34-mer DNA flap complex samples consistently gave
Io values ~10% larger that the FEN-1·34-mer DNA
flap complex samples, suggesting that these samples are aggregated, but
not severely. Despite the slight aggregation of the mutant complex, the
scattering data suggest that the D181A mutant binds and interacts
differently with the flap DNA substrate compared with wild-type
FEN-1.
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The 5'-nuclease domains of E. coli and Taq DNA
polymerases are functional homologs of human FEN-1 that possess two
Mg2+-binding sites. The amino acid sequences of 10 5'-nuclease domains from DNA polymerases in the polymerase I family and
viral nucleases were compared by Gutman and Minton (43). The results
showed six highly conserved sequence motifs containing 10 conserved
acidic residues. In earlier work, we extended this sequence comparison to eight additional sequences of XPG/FEN-1 nuclease family and confirmed that seven acidic amide residues are very conserved in all 18 sequences (35). According to the crystallographic structures of
Taq DNA polymerase (44) and T4 RNase H1 (45), these residues
(Asp-34, Asp-86, Glu-158, Glu-160, Asp-179, Asp-181, and Asp-233 in
human FEN-1 based on the superimposition between FEN-1 and T4 RNase H1)
cluster within a sphere of 7-Å radius around the metal ions. Some of
these residues ligate one Mg2+ ion, whereas others ligate
the second Mg2+ ion. Our mutagenesis work (34) further
confirmed that Asp-181 is a critical amino acid that ligates the
Mg2+ and is involved in the cleavage process of the flap
endonuclease activity only, but does not appear to affect DNA binding.
In this report, we found from SAXS that Asp-181 is actually important for proper binding of the DNA and is not only required for cleavage activity. In fact, proper binding proceeds enzymatic activity whereby
the proper orientation of the DNA in the catalytic site is required
before cleavage can ensue.
Measurement of the time course change in scattering after initiating
cleavage activity by adding magnesium to FEN-1·34-mer DNA flap
complex samples required slowing down the kinetics. Typically, for the
protein and substrate concentrations used in this report, activity
rates are such that it would require only minutes for DNA cleavage
completion, too fast to monitor with our scattering instrument, which
requires at least 30 min of measurement time to obtain good enough
statistics for calculating extrapolated Io values.
Addition of monovalent ions is know to slow kinetics for this system by
~100-fold (4); therefore, we added 100 mM NaCl and
performed measurements at the low temperature of 10 °C. After
initiating activity, we observed a decrease in the scattering intensity
as shown in Fig. 7a. The
decrease in scattering intensity continued for up to 6 h, after
which the profiles remained constant. When activating nuclease activity by adding Mg2+, we expect a decrease in
Io scattering if the activated protein and the DNA
components dissociate after DNA cleavage. Assuming complete
dissociation of the components after cleavage, we can write the total
scattering in terms of each species in solution as follows (Equation 2),
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(Eq. 2)
|
where ssDNA and dsDNA are single- and doubled-stranded DNA,
respectively. No scattering cross-term would be present between the
species in solution. It is possible that the protein remains bound to
either the single- or double-stranded DNA fragment after cleavage, in
which case, we would have the total scattering given by Equation 3,
|
(Eq. 3)
|
or Equation 4.
|
(Eq. 4)
|
The protein·DNA complex terms in Equations 3 and 4 would include
a scattering cross-term as similarly expressed in Equation 1. From
Equations 1-4, we would expect 0, 43.9, 32.5, and 15% decreases in
Io scattering, respectively. The "apparent"
Io scattering decreased by ~24-29% for two
independent measurements. This apparent decrease was determined by
comparing the first measurement made within 1 h of activation and
a final measurement made after 6 h of activation after which no
further decrease in Io was observed. At first
glance, this measured decrease in intensity appears most consistent
with FEN-1 remaining bound to the single-stranded portion of the DNA
fragment after cleavage. However, because our measurements were for 30 min, the initial t = 0 measurement is underestimated. A
crude estimate on the order of magnitude of the error in
underestimating the t = 0 measurement can be made by
assuming that the reaction is completed by 6 h and that it proceeds exponentially. A nonlinear least-squares "best" fit to a
plot of Io with respect time can be made using the form Io = A + Bexp(
t/t'),
where A and B are constants to be determined in
the fit and 1/t' is the reaction rate, also to be determined
in the fit. We assume a considerably larger error for the earliest
measurement (average t = 15 m) relative to the
later measurements and iterate the fit by successively replacing the
earliest measurement until the fit converges and then extrapolate to
t = 0. Using this simple and crude approximation gives
the exponential fit to the Io data as shown in Fig.
7b. We find that, under the conditions of this time course
experiment, Io at t = 0 is
underestimated by at least 21%. Taking this into account, the
correction in the decrease in Io would then be
~37-42% due to nuclease activity, more consistent with total
dissociation of the protein from its DNA substrate. It is worth
pointing out that, regardless of error estimates, we can conclude with
certainty that the protein is not bound solely to the remaining nicked
double-stranded DNA component since the measurement at
t = 0 can be only underestimated, not overestimated.
Similar measurements were made after Mg2+ was added to
D181A·34-mer DNA flap complex samples. In this case, no change in the
scattering intensity with respect to time was observed.

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|
Fig. 7.
Time course SAXS measurement showing
Mg2+ activation of the FEN-1 activity.
Mg2+ was added to the FEN-1·34-mer DNA flap complex (1:1)
at t = 0 h, and scattering profiles were record at
intervals for a 30-min duration. Profile measurements at
t = 0 (measurement between 0 and 30 min; black
circles) and at t = 6 h (gray squares) are
shown in a. We added 100 mM NaCl to the buffer
and performed measurements at 10 °C to slow down the nuclease
activity rate. After 6 h, Io did not decrease
any further. In b, we show Io
(black circles) versus time fitted to an
exponential function of the form Io = A + Bexp( t/t',
where A and B are constants to be determined in
the fit and 1/t' is the reaction rate, also to be determined
in the fit. The fitting procedure was iterative so as to account for
the underestimation of the extrapolated t = 0 measurement. Note the underestimation of the first measurement, which
lies below the iterative exponential fit.
|
|
FEN-1 nuclease is critical in the maintenance of genome stability and
mutational avoidance. Yeast null mutants displayed a unique mutational
spectrum derived from the failure of RNA primer removal during the
lagging strand DNA synthesis. The SAXS study reported here provides a
global structure of this unique DNA replication and repair enzyme and
confirms the interaction of enzyme/flap DNA substrate/Mg2+
cofactor. It also suggests the critical role of Asp-181 in the enzyme/flap DNA substrate interaction to ensure a proper conformation and a cleavage-ready status of the enzyme. A time course change in the
scattering profiles arising from magnesium activation of the
FEN-1·DNA flap substrate complex is consistent with the enzyme being
freed from both the single- and double-stranded DNA product portions
after cleavage. Given the critical role of FEN-1 in DNA replication and
repair, this study illustrates initial understanding of molecular
dynamics common to the structure-specific nuclease family that are
central to processes influencing genome stability and early events that
modulate cancer susceptibility and tumorigenesis.
 |
ACKNOWLEDGEMENTS |
We are indebted to Dr. Jill Trewhella for
providing beam time on the small-angle x-ray scattering station at Los
Alamos National Laboratory (Los Alamos, NM). We give special thanks to
Dr. S. V. Santhana Mariappan (Los Alamos National Laboratory) for
advise on DNA substrate design, NMR analysis for the synthesized flap DNA substrate, and proofreading. We thank Dr. John Nolan for providing us with flap DNA-beads needed for the flow cytometry assay used to test
FEN-1 activity and David Eckhart for assistance in FEN-1 protein expression.
 |
Note Added in Proof |
During review of the manuscript, two
crystallographic structures of archaebacterial FEN-1s have been
published (Hosfield, D., Mol, C. D., Shen, B., and Tainer, J. A. (1998)
Cell 95, 135-146; Hwang, K. Y., Baek, K., Kim, H.-Y.,
and Cho, Y. (1998) Nat. Struct. Biol. 5, 707-713).
 |
FOOTNOTES |
*
This work was supported by the Integrated Structural Biology
Resource Program at Los Alamos National Laboratory, by Los Alamos National Laboratory Directed Research Development Grants 94205 (to
M. S. P.) and 95623 (to G. A. O.), by Department of Energy Grant
KP1104-010 (to M. S. P.) and by National Institutes of Health Grant
CA73764 (to B. S.).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.
§
Present address: Dept. of Cell and Tumor Biology, City of Hope
National Medical Center and Beckman Research Inst., Duarte, CA 91010.
To whom correspondence should be addressed: Oklahoma State
University College of Veterinary Medicine, 110 Veterinary Medicine, Stillwater, OK 74078. Tel.: 405-743-1887; E-mail:
olah{at}okstate.edu.
The abbreviations used are:
FEN-1, flap
endonuclease-1; SAXS, small-angle x-ray scattering; Rg, radius of gyration; Rc, radius of gyration of cross-section; Io, forward
scatter; P(r), vector distribution function; dmax, maximum linear dimension.
 |
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