From the
In this study, we have identified a protein in Escherichia
coli that specifically binds to double-stranded DNA containing a
single-stranded gap of one nucleotide. The gap-DNA binding (GDB)
protein was purified to apparent homogeneity. The analysis of the
amino-terminal sequencing of the GDB protein shows two closely related
sequences we identify as the
DNA transactions such as general and site-specific
recombination, replication, and repair imply the formation of
intermediates that contain single-stranded nicks or short gaps
(1, 2, 3, 4) . For example, the altered
bases in DNA are often excised by DNA glycosylases leading to the
formation of abasic sites. Such sites are also generated by the
nonenzymatic hydrolysis of sugar-base bonds and as a consequence of
exposure to ionizing radiation
(5, 6) . Abasic sites in
DNA are subjected to rapid incision and repair by the base excision
repair (BER)
Single-stranded nicks and gaps in duplex DNA are also
representative of a class of problems in nucleic acids structure.
Several studies have shown that the presence of nicks and short gaps
results in an increased flexibility of DNA at these sites
(10, 11, 12) . Thus, the formation of BER and
nucleotide excision repair intermediates in DNA may induce structural
changes that might be specifically recognized by proteins in the cell.
Indeed, these DNA intermediate structures represent potential weakness
points in the DNA chain, which may require transitory interactions with
putative ``DNA chaperones''
(13) . Such proteins might
facilitate the access at these sites to DNA repair enzymes. In
eukaryotes, at least one protein, the poly(ADP-ribose) polymerase
(PARP) is known to be able to bind specifically DNA strand breaks
(14) . Recent studies demonstrated that through its nick-DNA
binding activity, PARP can modulate DNA repair
(15, 16, 17) . Thus, since PARP is absent in
prokaryotic cells, bacteria may possess protein factors that recognize
such DNA structures.
In this study, we have attempted to identify
such protein(s) in E. coli. In this aim, we employed an
electrophoretic mobility shift assay to reveal high affinity complex
formation between some E. coli proteins and a 30-mer DNA
oligonucleotide duplex containing a single nucleotide gap. Using this
assay, we purified a protein that was determined to be the HU protein.
HU is a low molecular weight, thermostable histone-like protein that
forms heterodimers that bind to linear DNA fragments every 9 base pairs
regardless of their sequence or length
(18, 19, 20, 21, 22, 23) .
Moreover, in vitro and in vivo studies have also
shown that HU may be involved in cellular DNA processes such as DNA
transposition
(24, 25) , DNA inversion
(26, 27) , and replication
(28, 29, 30) . The ability of HU to bind to DNA
containing nicks or gaps, first described in this study, may be related
to these biological functions.
Fig. 4
shows that purified GDB protein and HU protein form a
single retardation complex with the [1 nt-gap] probe (C1
complex) that migrates at the same position ( lanes 2 and 3). The identification of HU as the GDB protein
purified in this study was confirmed using a mutant of E.
coli, which is defective in HU protein. Indeed, the GDB protein is
not detected in a crude extract from an E. coli mutant strain
( hupAB), where hupA and hupB genes, coding
for
In this study, we have purified and characterized a GDB
protein of E. coli. Together, the amino-terminal sequence and
the properties of the GDB protein indicate that this protein is the HU
protein. Until recently, it has been considered that HU binds DNA in a
nonspecific manner (see Introduction). In this work, we show that HU
recognizes specifically 30-mer double-stranded oligonucleotides
containing a single nick or a gap of one or two nucleotides. Under high
salt conditions used (200 mM KCl), the affinity for the
[1 nt-gap] DNA is at least 100-fold higher than for the
unmodified [C/G] DNA. The affinity of HU increases as the
size of the gap increases. Furthermore, the complex C1 formed between
HU and DNA containing a nick or a gap migrates faster than the complex
C1` formed between HU and unmodified DNA. This difference in
electrophoretic mobility may reflect structural differences between the
low and the high affinity complexes. Recent studies have shown that
nick or gap in DNA fragments result in an increased flexibility and in
the formation of V-shaped structure
(11, 12) . These
results are in favor of the recognition by HU of a DNA structure
resulting from the presence of a nick or a gap in the oligonucleotide
used. However, the recognition of the free ends of the gap by HU cannot
be excluded.
Our results could be compared with those of Pontiggia
et al. (43) and Bonnefoy et al. (23) showing that HU has a high affinity for cruciform and
bulged DNAs. The common feature between these structures and those that
DNA fragments containing a nick or gap may adopt is the possible
occurrence of a sharp angle
(10, 11, 12, 30) . The angle value seems
to be a critical parameter to explain this HU preferential binding.
Indeed, Pontiggia et al. (43) show that the HU
affinity for cruciform DNA ( K
In addition to DNA
packaging function, HU is involved in several DNA processing events
such as the bacteriophage Mu transposition
(24) , the Tn10
transposition
(25) , and the gene inversion
(27) . Hwang
and Kornberg
(30) have also demonstrated that HU plays a role
in the initiation of the DNA replication. In this work, we show that HU
binds tightly to BER intermediates in vitro, suggesting that
HU may play a role in DNA repair. In vivo, the putative role
of HU would at first consist in the stabilization of DNA structures
containing short single-stranded regions, protecting it from further
degradation by endogenous nucleases. Then, local DNA conformational
changes occurring through HU binding may contribute to the formation of
an active protein-DNA complex by modifying the DNA accessibility for
other proteins such as the DNA polymerase I. The hypothesis suggesting
that HU can modify the binding property of other cellular proteins is
consistent with previous studies showing that HU can modulate specific
DNA binding of regulatory proteins such as cyclic AMP receptor protein
(44) , integration host factor
(45) , and LexA repressor
(46) . In eukaryotes, a similar function has been proposed for
the PARP. Thus, Satoh and Lindahl
(16) have shown in a human
cell-free system that unmodified PARP binds tightly to
We thank Dr. J. Rouvière-Yaniv for the kind
gift of HU protein and bacterial strains. We thank J. P. Le Caer for
the determination by microsequencing of the amino-terminal sequence of
the homogeneous GDB protein. We are grateful to Dr. C. Paoletti for
constant and warm encouragement. We also thank Drs. F. Culard, T. R.
O'Connor, and D. Touati for helpful discussions and critical
reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
subunits of the HU
protein. Furthermore, the GDB protein is not detected in the crude
extract of an E. coli double mutant strain hupA
hupB that has no functional HU protein. These results led us
to identify the GDB protein as the HU protein. HU binds strongly to
double-stranded 30-mer oligonucleotides containing a nick or a
single-stranded gap of one or two nucleotides. Apparent dissociation
constants were measured for these various DNA duplexes using a gel
retardation assay. The K
values were 8 nM for the 30-mer duplex that contains a
nick and 4 and 2 nM for those that contain a 1- or a
2-nucleotide gap, respectively. The affinity of HU for these ligands is
at least 100-fold higher than for the same 30-mer DNA duplex without
nick or gap. Other single-stranded breaks or gaps, which are
intermediate products in the repair of abasic sites after incision by
the Fpg, Nth, or Nfo proteins, are also preferentially bound by the HU
protein. Due to specific binding to DNA strand breaks, HU may play a
role in replication, recombination, and repair.
(
)
pathway in both prokaryotes and
eukaryotes
(7, 8) . Their repair results from the
excision of a sugar phosphate followed by the insertion of one
nucleotide by a DNA polymerase and sealing of the nick by the DNA
ligase
(7) . Thus, in the majority of BER events, a
single-stranded gap of one nucleotide is formed as a repair
intermediate in duplex DNA. Larger gaps are also generated by the
Escherichia coli nucleotide excision repair that induces the
release of a single-stranded dodecamer containing the damaged site
(9) .
Enzymes, Bacterial Strains, and Preparation of
Bacterial Crude Lysates
Fpg (Fapy-DNA glycosylase), Nth
(endonuclease III), Nfo (endonuclease IV), and Ung (uracil-DNA
glycosylase) proteins were purified from E. coli overproducing
strains using procedures already described. The HU protein used for
comparison with the GDB protein is a kind gift from Dr. J.
Rouvière-Yaniv (IBPC, Paris). All the strains used were derived
from E. coli K12. The BH20 strain is isogenic with AB1157
except for fpg-1::Kan(31) . The JR 766
strain is isogenic with the E. coli C600 strain except for
hupA::Kan
and hupB::Cm
(32) . Cell cultures and bacterial crude extracts were
carried out as described by Boiteux et al. (33) .
DNA Oligonucleotides
High pressure liquid
chromatography-purified oligonucleotides were purchased from GENSET
(Paris). The sequences used are reported in Fig. 1.
Phosphorylation at the 5`-end of the oligonucleotides was performed
using T4 polynucleotide kinase and [-
P]ATP
(3000 Ci/mmol, Amersham Corp.) or cold ATP as described by Castaing
et al. (34) . Single-stranded 30-mer oligonucleotides
were annealed to complementary sequences to produce a control duplex
(Fig. 1, [C/G]) or duplexes that contain
either a nick ( [nick]) or single-stranded gaps of
one or two nucleotides ( [1 nt-gap]) and ( [2
nt-gap]), respectively (Fig. 1).
Figure 1:
Oligonucleotides used as DNA probes in
this study. An abasic site is shown as dr, a
5`-P-labeled strand is shown as p*, and
phosphorylation at the 5`-end with cold ATP is shown as p. An
arrow indicates the nick position in
[nick].
To yield
intermediate products of base excision repair, a 30-mer
5`-P-labeled single-stranded oligonucleotide containing a
uracil residue was treated with the Ung protein for 1 h at 37 °C
(35) . After this treatment, more than 95% of the uracil
residues were excised and replaced by an abasic site. The resulting
oligonucleotide was annealed with a complementary sequence containing a
guanine opposite the abasic site to produce the duplex (Fig. 1,
[dr/G]). The initial single-stranded oligonucleotide
containing a uracil was annealed with the same complementary sequence
to obtain a duplex ( [U/G]) (Fig. 1). To
prevent
elimination at the abasic site, the
( [dr/G]) duplex was stored in citrate buffer (SSC
5) at
20 °C and freshly thawed before use.
Bandshift Experiments and GDB Protein
Assay
The assay (24 µl, total volume) contained 25
mM Hepes/KOH, pH 7.6, 100 mM KCl, 5 mM
NaEDTA, 5 mM
-mercaptoethanol, 6% glycerol,
0.2 mg/ml poly(dI-dC), 5,000-10,000 cpm of
5`-
P-labeled double-stranded oligonucleotide probe
(50-100 pM), and 5-15 µg of proteins from
bacterial crude lysate or 5 µl of column fraction. When indicated,
an unlabeled oligonucleotide competitor (2.4 nM) was added
prior to the addition of the crude lysate. The binding reaction was
performed for 10 min at 20 °C. Then, samples were loaded onto a
non-denaturing 10% polyacrylamide gel for electrophoresis according to
Castaing et al. (34) . The gels were then fixed in a
10% acetic acid, 10% methanol solution (v/v), dried on 3MM Whatman
paper, and exposed for autoradiography to Kodak X-AR film at
70
°C. The specific binding of the [1 nt-gap]
oligonucleotide was used as an activity assay to purify the GDB protein
of E. coli.
Determination of Apparent Dissociation
Constants
The apparent dissociation constants
( K) were determined by
the gel retardation method. The incubation mixtures were as above,
except that assays were performed at 200 mM KCl without
competitor poly(dI-dC) and in the presence of 1500 cpm (15 pM)
of labeled oligonucleotide probes. Assuming a stoichiometry of 1:1 for
the complex between the heterodimeric HU protein and the DNA probe, the
K
values are calculated directly from the
electrophoresis data, as previously described by Castaing et al. (34) .
Purification of a GDB Protein from E.
coli
1 liter of LB broth medium containing 40 µg/ml
kanamycin was inoculated with 10 ml of an overnight culture of the
E. coli strain BH20. Bacteria were grown at 37 °C until
A= 2.0. Cells were harvested, washed,
and resuspended in buffer A (25 mM Tris/HCl, pH 8.2, 5
mM
-mercaptoethanol, 5% glycerol, and 1 mM
Na
EDTA) containing 250 mM NaCl. The cell
suspension was supplemented with 1 mg/ml lysozyme, incubated for 10 min
at 37 °C, and frozen for 10 min at
70 °C. This
freezing-thawing cycle was repeated twice. The lysate was then
centrifuged at 35,000 r.p.m. for 1 h at 4 °C. The resulting
supernatant was the crude extract (fraction I). Fraction I was loaded
on a QMA anion exchange column (Waters Acell), washed, and equilibrated
in the buffer A containing 250 mM NaCl. The active fraction
was eluted in the flow-through volume (fraction II). Fraction II was
then heated at 100 °C for 30 min and centrifuged at 10,000 r.p.m.
for 15 min. The active GDB protein was recovered in the supernatant
(fraction III). Fraction III was dialyzed against buffer A containing
25 mM NaCl and applied to a second QMA-Acell column, washed,
and equilibrated in buffer A containing 25 mM NaCl. The GDB
activity was recovered in the flow-through volume (fraction IV).
Fraction IV was adjusted to pH 7.2 and loaded onto a Phospho-Ultrogel
column (Sepracor, MA). The column was eluted with a linear NaCl
gradient (25-600 mM). The active fractions eluted
between 300 and 400 mM NaCl (fraction V). Fraction V was
dialyzed against buffer A (pH 7.2) containing 25 mM NaCl and
loaded onto a DNA-cellulose column (Sigma). This column was eluted with
a linear NaCl gradient (25-500 mM). The GDB protein
eluted at 250 mM NaCl (fraction VI). Fraction VI (1 mg of
protein) was dialyzed and stored at 0 °C. Amino-terminal
microsequencing was performed as described by Le Caer and Rossier
(36) .
Identification and Purification of an E. coli GDB
Protein
The goal of this study was to identify GDB proteins
in E. coli. To identify such proteins, we used a gel
retardation assay with 30-mer DNA probes either fully double-stranded
[C/G] or containing a single-stranded region of one
nucleotide [1 nt-gap] (Fig. 1). Fig. 2shows the
electrophoretic mobility of DNA probes [1 nt-gap] or
[C/G] when they are mixed with E. coli crude
extract. Several retardation complexes are formed including a well
defined C1 complex and other complexes ( Cn) migrating near the
top of the gel ( lanes 2 and 6). The C1
complex, however, is only observed with the [1 nt-gap] probe
( lane 6). This complex is not detected with the
unmodified control [C/G] duplex ( lane 2).
Furthermore, competition experiments with cold [C/G] duplex
for the binding of the [1 nt-gap] duplex shows that only C1
is conserved ( lane 8). These results strongly suggest
that the C1 complex results from the specific interaction between the
[1 nt-gap] probe and a cellular factor. Control experiments
show that C1 complex is due to a heat stable cellular factor, since the
incubation of crude extract at 100 °C for 30 min does not affect
the GDB activity ( lane 7). In addition, the GDB
activity is abolished after treatment of the extract with proteinase K
( lane 10). These results show that E. coli possesses a thermostable protein that specifically binds a
double-stranded DNA fragment that contains a single-stranded region of
one nucleotide.
Figure 2:
Identification of an E. coli GDB
activity. Reaction mixtures containing either 5`-P-labeled
[C/G] or [1 nt-gap] duplex (50 pM) were
incubated with E. coli crude extract (10 µg of proteins)
for 10 min at 20 °C. All reactions contained competitor poly(dI-dC)
and, when indicated, an additional 40-fold excess of cold
[C/G] or [1 nt-gap] duplex. When indicated, the
E. coli extracts were boiled for 30 min. The gel retardation
assay was performed using a 10% polyacrylamide gel under non-denaturing
conditions at 4 °C (for details see ``Materials and
Methods''). C1 corresponds to the specific HU-[1
nt-gap] complex, and Cn corresponds to other complexes
obtained with any DNA probes.
The specific binding of the [1 nt-gap]
probe, detected by the C1 complex, was used as an activity assay to
purify the GDB protein of E. coli. Purification steps are as
described under ``Materials and Methods,'' and the final
fraction VI shows a single protein band on SDS-polyacrylamide gel
electrophoresis with an apparent molecular mass of 7 kDa
(Fig. 3).
Figure 3:
Analysis of the GDB protein purification
fractions using SDS-polyacrylamide gel electrophoresis. The E. coli GDB protein was purified as described under ``Materials and
Methods.'' Active fractions (I-VI) from each purification
step were analyzed in a 16% polyacrylamide gel in the presence of SDS.
The amount of protein loaded on each gel was as follows: lane I, 30 µg; lane II, 20 µg;
lane III, 15 µg; lane IV, 10
µg; lane V, 5 µg; and lane VI, 1 µg. In lane M, the molecular
mass markers were phosphorylase b (94 kDa), bovine serum
albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa),
trypsin inhibitor (20 kDa), and -lactalbumin (14 kDa). In lane m, the molecular mass markers were intact myoglobin (17.2
kDa) and its partial cyanogen bromide cleavage product, I + II
(14.6 kDa), I (8.24 kDa), II (6.38 kDa), and III (2.56
kDa).
GDB Protein Is the HU Protein
The
homogeneous GDB protein contained in fraction VI was transferred onto
Problot membrane, and its amino-terminal sequence was determined. We
obtained the sequence MNK(T/S)QLID(V/K)IAE(G/K)A(E/D), where the amino
acids between brackets correspond to a double signal in a single round
of degradation. The results suggest the presence of two peptides with
closely related sequences. A computer search for alignment of these two
sequences indicated a perfect identity with the first 15 amino acids of
the and
subunits of the E. coli HU protein,
respectively
(20) . These results strongly suggest that the GDB
protein isolated in this study is the HU protein. Moreover, earlier
studies showed that HU is heat stable
(18) . Like the HU
protein, the GDB protein activity is still detected in crude extract
after boiling for 30 min (Fig. 2, lane 7).
and
HU subunits, respectively, were inactivated
( lane 6). In contrast, crude extract from the
isogenic wild type strain possesses the thermostable GDB activity
( lanes 4 and 5). Furthermore, it was
possible to complement a hupAB crude extract for the GDB
activity by the addition of the purified GDB protein in the binding
mixture ( lane 7). Finally, the GDB protein and HU
form the same retardation complexes in the presence of the control
30-mer oligonucleotide (data not shown). These results led us to
conclude that the purified GDB protein from E. coli and the HU
protein are the same protein.
Figure 4:
Identification of the purified E. coli GDB protein as the HU protein. The purified GDB protein (30
nM), the purified HU protein (30 nM), or crude
extracts from various E. coli strains (10 µg) were
incubated under the experimental conditions described in Fig. 2. The
E. coli extracts were derived from the C600 strain
( wt), which yields the wild type HU protein, and from the
mutant JR 766 strain ( hupAB), isogenic with the C600 strain
but being unable to produce an active HU protein. See Fig. 2 for
experimental binding conditions and for the definition of C1 and Cn.
HU Protein Has a High Affinity for DNA Duplexes
Containing Single-stranded Nicks or Gaps
The HU protein was
initially identified in E. coli as a DNA binding protein able
to bind single- and double-stranded DNA molecules as well as
ribonucleic acids in a non-sequence-specific way
(22, 37, 38) . Here, we show that HU protein
binds specifically DNA that contains a single-stranded region of one
nucleotide. To quantify this specificity, we have determined by gel
retardation assay the apparent dissociation constant
( K) for 30-mer DNA
oligonucleotides containing either no modification
( [C/G]) or a nick ( [nick]) or a
gap of one ( [1 nt-gap]) or two ( [2
nt-gap]) nucleotides (Fig. 5). Determination of
dissociation constants was carried out at 200 mM KCl to use
more stringent conditions for DNA binding. Using the unmodified
double-stranded oligonucleotide [C/G] and within the protein
concentration range used (0-10 nM), only limited
formation of a C1` complex was observed (Fig. 5 A). About
200 nM HU is needed to bind 50% of the total [C/G]
DNA in three complexes (data not shown). Complex C1` corresponds to the
first complex formed between one HU heterodimer and double-stranded
DNA. In contrast, only 10 nM HU is sufficient to bind the
major part of the three other oligonucleotides containing DNA strand
breaks in the C1 complex (Fig. 5). The HU affinity increases from
the [nick] with a K
= 8 nM (Fig. 5 B) to the [1
nt-gap] with a K
= 4 nM (Fig. 5 C) and to the
[2 nt-gap] with a K
= 2 nM (Fig. 5 D). These
K
values show that the
DNA binding increases as a function of the length of the
single-stranded region. It should be noted that the C1 complex migrates
faster than the C1` complex (Fig. 5). This result may be due to
structural differences contained in the complex formed between HU and
unmodified DNA (C`1) and complexes formed between HU and DNA containing
a nick or a short single-stranded gap (C1).
Figure 5:
Apparent dissociation constants
( K) of HU for DNA duplexes containing
single-stranded breaks. Reaction mixtures containing radiolabeled
duplexes (20 pM [C/G], [nick], [1
nt-gap], or [2 nt-gap] as shown in Fig. 1) were
equilibrated in 10 µl final volume at 20 °C for 10 min with the
indicated HU protein concentrations. Binding conditions and apparent
dissociation constant measurements are described in the text and under
``Materials and Methods.'' C1 complex is defined in Fig. 2,
and C1` complex corresponds to the first complex formed between HU and
the unmodified DNA.
Intermediate Products of Base Excision Repair Are
High Affinity Ligands for the HU Protein in Vitro
An abasic
site is a common DNA repair intermediate in the course of BER
(8) . Fig. 6summarizes the different DNA intermediates
generated by enzymes that incise DNA at AP sites in E. coli (35, 39, 40, 41) . These various
structures are potential ligands for the HU protein as shown in this
work. To explore this question, a 30-mer single-stranded
oligonucleotide containing one uracil residue was treated with an
excess of the Ung protein and then annealed with its complementary
strand to generate a double-stranded oligonucleotide containing a
unique AP site opposite a guanine residue (Fig. 1,
[U/G] and [dr/G]). The
[dr/G] duplex was then incubated with several AP nicking
enzymes such as Fpg, Nth, or Nfo proteins to yield the various repair
intermediates (Fig. 6). The various oligonucleotides were
incubated with HU under the same conditions, and the reactions were
analyzed by bandshift assays (Fig. 7). As observed with the
[C/G] duplex, HU is able to recognize [U/G] and
[dr/G] duplexes to yield a weak amount of the C1` complex
( lanes 2 and 7). This result indicates that
an abasic site is not a preferential ligand for the HU protein compared
to unmodified DNA. Conversely, nicking of abasic sites at the 5`-side
by Nfo ( lane 13), at the 3`-side by Nth ( lane 11), and at both 3`- and 5`-sides by Fpg ( lane 9) or by [Nfo/Fpg] or [Nth/Nfo]
combinations ( lanes 15 and 17) generate high
affinity ligands for the HU protein. The resulting complexes migrate at
the same position as the C1 complex corresponding to the HU-[1
nt-gap] complex ( lane 19). Fig. 7also
shows that the Fpg protein formed an additional complex called C2 with
unmodified [U/G] probe ( lane 3). The
intensity of C2 increased after excision of the uracil residue
( lanes 8 and 14). This observation may
reflect the relative affinities of the Fpg protein for DNAs containing
no modification or abasic sites
(34, 42) . These last
results also suggest that Fpg protein retains a high affinity for its
terminal product (a gap of one nucleoside; see Fig. 6). When both
HU and Fpg are present in the assay, we observe the C1 and C2 complexes
simultaneously indicating a competition between these two proteins for
the gap generated by Fpg (Fig. 7, lanes 9 and
16).
Figure 6:
Schematic representation of BER
intermediates after incision of an AP site by repair endonuclease in
E. coli. AP sites are specifically incised by two classes of
repair activities: (i) AP lyases that catalyze the cleavage at 3`-side
of AP sites by elimination (Nth protein) or at both 3`- and
5`-sides of AP sites by
elimination (Fpg protein) and (ii)
AP endonucleases that cleave hydrolytically at 5`-side of AP sites (Xth
and Nfo proteins). The resulting baseless sugar phosphate residue (dRp)
may be removed by a DNA deoxyribophosphodiesterase (dRpase) like RecJ
and Fpg proteins, by a 5`
3` exonuclease or by the combined
action of both classes of endonucleases. The one nucleotide gap is the
most frequent final product, whereas larger gaps may be also produced.
These gaps are finally filled in by a DNA polymerase, and the resulting
nicks are sealed by a DNA ligase.
Figure 7:
Preferential binding of HU to DNA repair
intermediates. The indicated 5`-P-labeled DNA probes are
described in Fig. 1. The [U/G] probe was incubated in the
presence of the Ung protein yielding the [dr/G] DNA product
that contains an AP site. The resulting [dr/G] probe was
further incubated in the presence of an excess of enzymes that incise
DNA at AP sites yielding various DNA repair intermediates. When
indicated, HU was added to the reaction mixture without elimination of
the AP nicking enzyme. Incubation was carried out for 10 min at 25
°C. Then, samples were loaded onto a non-denaturing gel and
analyzed as before. The C1 and C1` complexes are defined in Fig. 2. The
C2 complex contained the Fpg protein and any of DNA duplexes.
=
10
to 10
M) is
significantly higher than the one found for a 6-nucleotide bulge DNA
( K
= 3
10
M). These authors suggest that the angle produced by the
A6 bulge may not be optimal for the HU recognition. Bonnefoy et al. (23) propose that this optimal value might be 60° as
found in the X-shaped conformation of the cruciform DNA. These results
suggest a preferential HU binding for sharp bend or kink in DNA, which
by analogy is also suggested in our data.
-irradiated
DNA containing single-stranded breaks and that the
auto-poly(ADP-ribosyl)ation of the protein then provokes its release,
rendering these lesions accessible to DNA repair enzymes. By analogy
with PARP, the HU binding to DNA strand breaks may constitute a
cellular signal that facilitates DNA repair. According to this
hypothesis, the HU protein could modulate DNA repair in vivo.
This model relies, however, on in vitro experiments only and
requires testing in vivo.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.