©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
HU Protein of Escherichia coli Binds Specifically to DNA That Contains Single-strand Breaks or Gaps (*)

Bertrand Castaing (§) , Charles Zelwer (1), Jacques Laval , Serge Boiteux (¶)

From the (1) Groupe Réparation des Lésions Radio- et Chimio-Induites, URA 147, CNRS, Institut Gustave Roussy, 94805 Villejuif, Cedex, France Equipe de Cristallographie Biologique, Centre de Biophysique Moléculaire, UPR 4301, CNRS, rue Charles Sadron, 45071 Orleans, Cedex 2, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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 Kvalues 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.


INTRODUCTION

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)() 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) .

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.


MATERIALS AND METHODS

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::Kanand 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 Kvalues 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 NaEDTA) 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) .


RESULTS

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).

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 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 Kvalues 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.




DISCUSSION

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= 10to 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.

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 -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.


FOOTNOTES

*
This work was supported in part by CNRS and the European Community (EV5V-CT92-0223). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by fellowships from the CNRS and from the Fondation pour la Recherche Médicale. Present address: Equipe de Cristallographie Biologique, Centre de Biophysique Moléculaire, UPR 4301, CNRS, rue Charles Sadron, 45071 Orleans, Cedex 2, France.

To whom correspondence should be addressed. Tel.: 33-1-45-59-64-05; Fax: 33-1-46-78-41-20.

The abbreviations used are: BER, base excision repair; GDB, gap-DNA binding; PARP, poly(ADP-ribose) polymerase; AP site, apurinic/apyrimidinic site.


ACKNOWLEDGEMENTS

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.


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