HU Protein of Escherichia coli Has a Role in the Repair of Closely Opposed Lesions in DNA*

Mitsumasa Hashimoto {ddagger}, Barry Imhoff, Md Moshi Ali and Yoke W. Kow §

From the Department of Radiation Oncology, Emory University, Atlanta, Georgia 30303

Received for publication, April 15, 2003 , and in revised form, May 12, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ionizing radiation generates a wide spectrum of DNA damage, including strand breaks, abasic (AP)1 sites, base damages, and cross-links (12). The energy of ionizing radiation is not dispersed uniformly in the absorbing medium but as packets of energy along the tracks of the charge particles. Each deposition of energy can generate multiple ionization events that can lead to the generation of a unique class of lesion called clustered lesion (38). In addition, when ionizing radiation interacts directly with DNA, multiple DNA ionizations can occur at the site of interaction that can also lead to the formation of clustered lesion. Thus, both the direct and indirect action of ionizing radiation on DNA can lead to the generation of clustered lesions (38).

The nature of clustered lesions is complex. They can consist of a combination of DNA breaks, AP sites, and base damages, either present on the same or opposing strands of DNA (47). A double strand break can be considered as a clustered lesion consisting of two single strand breaks located in close proximity on opposing strands of DNA. Much effort has been directed to understanding the repair of ionizing radiation-induced lesions such as strand breaks, AP sites, and base lesions; however, the biological process involved in the repair of these lesions within a clustered site is poorly understood. Strand breaks, AP sites, and base damages are repaired predominantly via the base excision repair (BER) pathway (9, 10). When AP sites and base damages are processed via BER, intermediary strand breaks are generated. For clustered lesions consisting of DNA damage located on opposing strands of DNA, a double strand break can be generated if the excision of the second lesion is initiated before the first lesion is completely repaired. The formation of a double strand break can be more lethal and mutagenic than the original closely opposed lesions. To reduce the formation of double strand breaks during cellular processing of clustered lesions, it is likely that there are mechanisms within cells that help to reduce the probability of initiating the repair of a second lesion before the first lesion is fully repaired.

It is interesting to note that many of the DNA repair glycosylases that initiate the repair of base lesions and AP sites are intrinsically inhibited by nearby nicks. Escherichia coli endonuclease III and formamidopyrimidine N-glycosylase activities are inhibited by nicks that are in close proximity and opposite to the base lesion (1114). The observed inhibition could be due to the fact that a nick increases the flexibility of DNA around the remaining lesion. The increased flexibility could lead to a decrease in the affinity of these DNA glycosylases for lesions that are located close to a nick. However, prolonged incubation of DNA containing clustered damage with either endonuclease III or formamidopyrimidine N-glycosylase can still lead to the generation of double strand breaks. Because a nearby nick can only slow down the action of a repair glycosylase, it is therefore likely that additional mechanisms are necessary to further inhibit the action of DNA glycosylases so that generation of double strand breaks can be reduced during the repair of clustered lesions. We had show earlier that KU, a human double-stranded DNA-binding protein, can bind to a nick opposite dihydrouracil (DHU), leading to a significant inhibition of the human endonuclease III activity (15). The increased inhibition of human endonuclease III activity by KU protein will help to reduce the possibility of generating double strand breaks during the repair of closely opposed lesions. Furthermore, even if a double strand break is formed, KU can bind to the ends of a newly generated double strand break and tether the two ends together as a protein-DNA complex, preventing the possibility of mis-joining (15).

In this study, we showed that the E. coli DNA-binding protein HU also inhibits the activity of E. coli endonuclease III when a nick is present in close proximity and opposite to a base damage. In contrast, HU protein did not inhibit significantly the subsequent steps of the repair process, namely DNA repair synthesis and DNA ligation. Thus HU could potentially mediate the sequential repair of a clustered lesion consisting of closely opposed DNA damage. These data further suggest that DNA-binding proteins that have high affinity for nicks, such as poly (ADP-ribose) DNA polymerase, could also mediate similar processes in the cells.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA Substrates—All oligonucleotides were obtained from Operon and purified by polyacrylamide gel (15%) electrophoresis as described previously (16). Oligonucleotides were 5'-end-labeled with [{gamma}-32P]ATP (Amersham Biosciences) using T4 polynucleotide kinase, following the instructions provided by the supplier. 32P-labeled oligonucleotides containing DHU were annealed to the appropriate complementary strands at 1:1.5 ratios in a buffer containing 10 mM Tris-HCl, pH 7.5, 0.1 M NaCl, and 2 mM mercaptoethanol by heating the mixture to 90 °C and cooling down slowly to room temperature. The oligonucleotide duplexes in Sequence I were used in this study (Q represents dihydrouracil). Duplex N contains a nick and a DHU. The nick is two nucleotides 3' from DHU on the complementary strand. In contrast, duplex L contains only a single DHU.



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SEQUENCE I
 

To determine the effect of distances between the nick and DHU on the inhibition of endonuclease III activity by HU protein, the substrates were prepared as in Sequence II.



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SEQUENCE II
 

Duplex DNA containing a 5' flap was constructed by hybridizing a 5' 32P-labeled 33-mer and an unlabeled 16-mer with the complementary 30-mer. This will generate a duplex containing a 5'-labeled flap of 19 nucleotides long deriving from the unhybridized 5' sequences of the 33-mer. The sequence of the three oligonucleotides for preparing the flap DNA is as follows: 33-mer, 5'-ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC; 16-mer, 5'-CAGCAACGCAAGCTTG; 30-mer, 5'-GTCGACCTGCAGCCCAAGCTTGCGTTGCTG; and Sequence III, where (Fp) = flap, 5'-ATGTGGAAAATCTCTAGCA.



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SEQUENCE III
 

Enzymes and Proteins—E. coli endonuclease III was purified from an overproducing E. coli strains employing MonoS, MonoQ and phenyl-Sepharose column as described previously (17). Endonuclease V was purifed from an E. coli overproducing strain employing MonoS, MonoQ, and phenyl-Sepharose (18). A small amount of HU protein was initially obtained as a gift from Dr. Roger McMaken (John Hopkins University), and was later purified from E. coli strains overproducing HU{alpha} and HU{beta} subunits following published procedures (19). HU protein is an 18-kDa heterodimeric protein consist of HU{alpha} and HU{beta} subunits, each subunit has a molecular mass of 9 kDa (19). Briefly, hupA and hupB genes were PCR amplified from E. coli genomic DNA. The hupA gene was PCR-amplified using primers 5'-CCCCCCCCATATGAACAAGACTCAACTGATTGATGTAATT and 5'-CCCCCCCCTCGAGCTTAACTGCGTCTTTCCAGTCCTTGCCA, which introduce NdeI and XhoI restriction sites near the ends of the PCR fragment. Similarly, the hupB gene was PCR-amplified using primers 5'-CCCCCCCCATATGAAATAAATCTCAATTGATCGACAAGATT and 5'-CCCCCCCCTCGAGGTTTACCGCGTCTTTCAGTGCTTTACCT. Each of the PCR products were restricted with NdeI and XhoI, and ligated into pET22b(+) that was previously restricted with NdeI and XhoI, using T4 DNA ligase (16 °C for 16 h) and electroporated into E. coli BL21(pLysS), producing ampicillin-resistant colonies. The constructs thus generated (pHuA and pHuB) code for HU{alpha} and HU{beta} modified to contain six C-terminal histidines. BL21(pLysS) cells harboring either pHuA or pHuB were grown to 0.7 OD, and overproduction of HU{alpha} and Hu{beta} were achieved by the addition of 0.5 mM of isopropyl-1-thio-{beta}-D-galactopyranoside and continued to grow the cells for an additional 16 h at room temperature. HU{alpha} and Hu{beta} proteins were then purified individually using nickel-nitrilotriacetic acid columns, following instructions supplied by the manufacturer (Novagen). E. coli DNA polymerase I and E. coli DNA ligase were purchased from USB biochemicals.

Enzyme Assays—Endonuclease III activity was assayed in a standard reaction mix (10 µl) containing 0.1 M KCl, 10 mM Tris-HCl, pH 7.5, 50 fmol of labeled DNA substrates, and 20 fmol of endonuclease III. The reaction was performed at 37 °C for 10 min.

Nick translation catalyzed by E. coli DNA polymerase I was performed in a buffer solution (10 µl) containing 67 mM potassium phosphate, pH 8.0, 6.7 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 33 µM dATP, 33 µM dCTP, 33 µM dGTP, and 33 µM dTTP. Duplex N (see Sequence I) is composed of a 56-mer containing a DHU hybridized to two complementary DNA, a 21-mer and a 35-mer. The 35-mer was 5'-end-labeled with 32P and used as the primer for E. coli polymerase I-dependent nick translation DNA synthesis. The rate of formation of a labeled full-length product was used as an estimate for the E. coli polymerase I activity.

DNA ligation was also performed with duplex N in a ligation buffer (10 µl) containing 30 mM Tris, pH 8.0, 4 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 20 µM NAD+, and 5 µg of bovine serum albumin. In this case, the 21-mer was 5'-end-labeled to provide the 5'-phosphoryl group that is required for the ligation reaction. The rate of formation of a full-length, 5'-end-labeled 56-mer was used for estimation for the DNA ligase activity.

Endonuclease III, nick translation and DNA ligation reactions were stopped with 5 µl of a stop buffer containing 90% formamide, 10 mM EDTA, 0,.1% xylene, and 0.1% bromphenol blue. After the addition of a stop buffer, the reaction mixture was immediately heated at 90 °C for 10 min. A 5-µl sample was loaded onto a 12.5% denaturing polyacrylamide gel and electrophoresed at 2000 V for 1.5 h. The polyacrylamide gel was then dried and the amount of products formed was estimated by using the STORM PhosphoImager (Amersham Biosciences).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of HU Protein on Endonuclease III Activity—HU protein binds tightly to various DNA replication and recombination intermediate structures (2022). In addition, it has a high affinity for DNA containing nicks and small gaps. Castaing et al. (23) showed that HU bound to DNA containing one base gap generated by DNA glycosylases. These data suggest that HU might play a role in preventing the untimely exonuclease digestion of DNA in cells by inhibiting the binding of exonuclease III to nicks and small gaps. Kamashev and Rouviere-Yaniv (20) showed that inhibition of exonuclease III activity by HU protein was only observed with DNA containing nicks. However, HU protein did not protect intact linear DNA from exonuclease III digestion, even at very high HU protein concentrations (20). Earlier, we showed that the human KU70/80 complex inhibited the rate of removal of DHU by human endonuclease III (15). Inhibition was only observed when the DNA substrate contained a DHU that was close to and opposite a nick. Because HU protein also binds tightly to DNA containing a nick, it is thus expected that, like human KU protein, HU protein might also inhibit the endonuclease III activity. Fig. 1 shows the effect of increasing HU protein concentrations on the endonuclease III activity. Two DNA substrates were used in this experiment: duplex N, which contained a nick opposite and in close proximity to a DHU, and duplex L, which contains only a DHU. When duplex L was used as a substrate, increasing the amount of HU protein had no effect on the activity of E. coli endonuclease III (Fig. 1, panel A). However, when duplex N was used as the substrate, increasing the amount of HU protein led to a significant inhibition of the endonuclease III activity (Fig. 1, panel B). At 400 nM of HU protein, greater than 75% inhibition of endonuclease III activity was observed. This is comparable with the inhibition of E. coli exonuclease III activity by HU; at 400 nM HU inhibited ~50% of the exonuclease activity of exonuclease III on nicked plasmid DNA (20).



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FIG. 1.
Effect of HU on endonuclease III activity. Panel A, 20 fmols of duplex L were incubated with 10 fmols of E. coli endonuclease III and an increasing amount of HU in a standard endonuclease III buffer at 37 °C for 10 min. The amount of endonuclease III-induced nicks was measured by electrophoresis on a 12.5% denaturing polyacrylamide gel. Duplex L incubated with HU only ({diamondsuit}) or with endonuclease III and HU ({square}). Panel B, 20 fmols of duplex N were incubated with 10 fmols of E. coli endonuclease III and an increasing amount of HU in a standard endonuclease III buffer at 37 °C for 10 min. Duplex N was incubated with HU only ({diamondsuit}) or with endonuclease III and HU ({square}).

 

To determine the effect that distances between the nick and DHU have on the inhibitory effect of HU on endonuclease III activity, five additional substrates were prepared with distances between the nick and DHU varied from 2, 4, 6, and 8 nucleotides lengths. Fig. 2 showed that HU exerted its maximum inhibitory effect when the nick was 2 nucleotides from the base lesion DHU. At 400 nM of HU protein, 50% of endonuclease III activity was inhibited when the nick was 2 nucleotides away, either 3' or 5' from DHU. When the nick was 4, 6, and 8 nucleotides away from DHU, inhibition of endonuclease III activity by HU was observed to be about 35, 22, and 12%, respectively.



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FIG. 2.
Effect of distance between the nick and DHU on the inhibitory effect of HU on endonuclease III activity. 20 fmols of duplex DNA containing nicks that are 0, 2, 4, 6, and 8 nucleotides from DHU on the complementary strand were incubated with 10 fmols of E. coli endonuclease III and 400 nM of HU in a standard endonuclease III buffer at 37 °C for 10 min. The amount of endonuclease III-induced nicks was measured by electrophoresis on a 12.5% denaturing polyacrylamide gel. Under the reaction condition used, endonuclease III activity on control DNA (CON) was 14.8 fmols of nick per 10 min (100%).

 

Effect of HU on DNA Polymerase I Activity—To have sequential repair of closely opposed lesions, it is necessary that repair synthesis catalyzed by DNA polymerase I (Pol I) be carried out unimpeded on the DNA strand containing the newly generated nick, whereas the cleavage activity of endonuclease III on the opposing lesion is inhibited by HU protein. It is therefore expected that concentrations of HU protein that inhibit endonuclease III activity will have little effect on the activity of Pol I. The effect of increasing concentrations of HU protein on Pol I activity was examined by using duplex N with the 35-mer 5'-end-labeled with 32P. DNA synthesis, or DNA polymerase activity was measured by the rate of extension of the 5'-end-labeled 35-mer to the full-length 56-mer. Because E. coli Pol I contains both 3'-5' and 5'-3' exonuclease activity, the amount of Pol I used for this experiment was chosen by determining the amount of Pol I that will give a good rate of DNA synthesis but little degradation of labeled primer. Fig. 3 shows a titration of the amount of Pol I used in the competing reactions of primer extension and degradation. It was found that under the reaction conditions used, 0.01–0.05 units of Pol I gave an optimum rate of primer extension with little degradation of the primer (Fig. 3, lanes 6–8). However, higher concentrations of Pol I led to substantial degradation of the labeled 35-mer (Fig. 3, lanes 2–5). Based on these data, the effect of HU protein on the nick translation was studied with 0.01 units of DNA Pol I. Fig. 4 shows that increasing concentrations of HU protein (up to 400 nM) had little inhibitory effect on the Pol I nick translation-DNA synthesis activity (Fig. 4, panel A, lanes 5–8). Using 0.01 units of Pol I, a 10 min reaction converted 45% of the substrate to the expected 56-mer full-length product (Fig. 4, panel B). The extent of nick translation catalyzed by Pol I was little affected, even in the presence of 400 nM of HU protein (Fig. 4, panel B). Recent studies showed that in the presence of poly (ADP-ribose) DNA polymerase, the human polymerase {beta} favors a strand displacement synthesis (24). Because Pol I can also carry out strand displacement DNA synthesis, it is thus of interest to find out whether, in the presence of protein, Pol I can shift from nick translation to strand displacement DNA synthesis. To do this, both the 35-mer and the 21-mer of duplex N were 5'-end-labeled with 32P. Fig. 5 shows the time course of the reaction with Pol I. At an early time interval (1 min after the addition of Pol I), 400 nM of HU showed a slight inhibition on the rate of formation of the full-length 56-mer (panels A and B, lane 1). In the presence of 400 nM HU, the 1 min reaction generated predominantly primer extension products that are shorter than the full-length product (panel B, lane 1). However, as the reaction progressed, most of the extended products were extended to become full-length 56-mer (panel B, lanes 2–9). However, the rate of degradation of the 5' DNA strand (the labeled 21-mer) was not inhibited (the faster migrating species of all lanes of panels A and B). These data suggest that high concentration of HU only slightly slowed down the initial extension of the 35-mer and appeared to have little effect on the progression of the nick translation reaction. This was indicated by the rapid formation of the full-length product and the extent of the degradation of the labeled 21-mer. These data thus suggest that the presence of HU protein did not shift Pol I from the nick translation mode of DNA synthesis to strand displacement mode of DNA repair synthesis.



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FIG. 3.
Titration of E. coli DNA polymerase I activity. 20 fmols of duplex N containing a 5'-labeled 35-mer was incubated in a standard nick translation buffer with decreasing E. coli DNA Pol I concentration. The nick translation reaction was performed at 37 °C for 10 min. At the end of 10 min, the reaction mix was assayed with a denaturing 12.5% polyacrylamide gel for the formation of full-length nick translation product. Lane 1, control duplex N showing the 5'-labeled 35-mer (primer). Lane 2–8, duplex N was incubated with 1 unit (lane 2), 0.5 units (lane 3), 0.25 units (lane 4), 0.1 units (lane 5), 0.05 units (lane 6), 0.025 units (lane 7), and 0.01 units (lane 8) of Pol I.

 


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FIG. 4.
Effect of HU on DNA polymerase I activity. Panel A, DNA polymerase I activity was measured by its nick translation activity. 20 fmols of duplex N containing a 5'-labeled 35-mer was incubated in a standard nick translation buffer with 0.01 unit of E. coli DNA Pol I and an increasing amount of HU protein. The nick translation reaction was performed at 37 °C for 10 min. At the end of 10 min, the reaction mix was assayed with a denaturing 12.5% polyacrylamide gel for the formation of full-length nick translation product. Lanes 1–4, duplex N was incubated with 0 nM (lane 1), 100 nM (lane 2), 200 nM (lane 3), and 400 nM (lane 4) of HU protein. Lanes 5–8, duplex N was incubated with 0.01 unit of Pol I and 0 nM (lane 5), 100 nM (lane 6), 200 nM (lane 7), and 400 nM (lane 8) of HU protein. Panel B, nick translation assays performed in panel A were quantified using the Storm PhsophoImager (Amersham Biosciences). Duplex N was incubated with increasing amounts of HU ({square}) or with 0.01 unit of Pol I and increasing amounts of HU ({triangleup}).

 


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FIG. 5.
Effect of HU on the 5'-3' exonuclease activity of DNA polymerase I. 20 fmols of duplex N containing a 5'-labeled 35-mer and 5'-end-labeled 21-mer was incubated in a standard nick translation buffer with 0.01 unit of E. coli DNA Pol I and 400 nM of HU protein. The nick translation was assayed by the formation of a full-length 56-mer and the 5'-3 exonuclease activity by the formation of labeled nucleotide shorter than the 21-mer. Lane C, control duplex N containing the 5'-labeled 35-mer and 21-mer. Panel A, nick translation was performed in the absence of HU protein. Panel B, nick translation was performed in the presence of 400 nM HU protein. For both panels A and B, 1 min reaction (lane 1), 2 min reaction (lane 2), 3 min reaction (lane 3), 4 min reaction (lane 4), 5 min reaction (lane 5), 8 min reaction (lane 6), 10 min reaction (lane 7), 12 min reaction (lane 8), and 15 min reaction (lane 9).

 

In addition to being a DNA polymerase, Pol I is also a flap endonuclease that is capable of cleaving the 5' flap from a flap DNA structure (25). Flap DNA structures are thought to be formed on the DNA lagging strand during processing of Okazaki fragment (26). HU was shown to bind to flap DNA with high affinity (22). It is thus of interest to find out whether the flap endonuclease activity of Pol I is affected by HU protein. 5'-Labeled flap DNA was prepared by hybridizing 5' 32P-labeled 33-mer and unlabeled 16-mer with the complementary 30-mer, generating a duplex DNA having a 5'-labeled flap of 19 nucleotides long. We have shown earlier that both the E. coli endonuclease V and Pol I cleave the 5' flap at the second phosphodiester bond 3' to the flap junction (27), generating a 5'-end-labeled 20-mer. Fig. 6 showed that incubating the labeled 5' flap DNA with E. coli endonuclease V generated predominantly a 20-mer and a small amount of 19-mer, an observation that is consistent with the earlier findings (Ref. 26; Fig. 6, lane 5). Furthermore, increasing the amount of HU protein led to inhibition of the flap endonuclease activity of endonuclease V (Fig. 6, lanes 6–8). The inhibition of the flap endonuclease activity by HU is most likely the result of HU binding to the flap junction because HU binds tightly to a flap junction (Kd = 1.8 nM; Ref. 20), Similarly, incubating the flap DNA with Pol I led to the generation of a 5'-end-labeled 20-mer (Fig. 6, lane 9), and the flap endonuclease activity of Pol was also inhibited by HU protein (Fig. 6, lanes 10–12). In contrast, the 5'-3' exonuclease activity of Pol I was not affected by increasing the amount of HU protein (Fig. 5).



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FIG. 6.
Effect of HU on the flap endonuclease activity of E. coli endonuclease V and DNA polymerase I. Flap endonuclease activity of endonuclease V and Pol I were assayed as described under "Experimental Procedures." 20 fmols of flap DNA containing a 5'-end-labeled flap was incubated with increasing amounts of HU (lane 1,0nM; lane 2, 100 nM; lane 3, 200 nM; lane 4, 400 nM), with 5 ng of E. coli endonuclease V and increasing amounts of HU (lane 5, 0 nM; lane 6, 100 nM; lane 7, 200 nM; lane 8, 400 nM), and with 0.05 units of Pol I and increasing amounts of protein HU (lane 9, 0 nM; lane 10, 100 nM; lane 11, 200 nM; lane 12, 400 nM).

 

Effect of HU Protein on DNA Ligase Activity—It was shown earlier that KU 70/80 protein complex increases the ligation efficiency of human DNA ligase III (28). It is also interesting to note that HU is essential for DNA ligase to effectively ligate short DNA fragments into circles (29). Therefore, we expect that HU protein will have little or no effect on the rate of ligation catalyzed by E. coli DNA ligase. To examine this, duplex N was used as the ligation substrate, and the 21-mer was 5'-end-labeled with 32P. This will provide the 5' phosphate that is necessary for the DNA ligase reaction. At the two DNA ligase concentrations (0.1 and 0.01 units), HU protein showed little or no inhibitory effect on the extent of ligation (Fig. 7, panel A, lanes 5–12). In the absence of HU, a 10 min reaction with 0.1 or 0.01 units of E. coli DNA ligase produced 74.6 and 6.5% of ligation, respectively (Fig. 7, panel B). HU protein showed little or no inhibitory effect on the extent of ligation even at 400 nM of HU, a concentration that led to greater than 75% inhibition of endonuclease III activity (Fig. 7, panel B).



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FIG. 7.
Effect of HU on E. coli DNA ligase activity. Panel A, 20 fmols of duplex N containing a 5'-labeled 21-mer was incubated in a standard ligation without DNA ligase (lanes 1–4), with 0.1 units of DNA ligase (lanes 5–8), and with 0.01 units of DNA ligase (lanes 9–12). DNA ligase activity was assayed by the formation of a full-length 56-mer. Varying amounts of HU were added to each reaction mix: 0 nM (lanes 1, 5, and 9); 100 nM (lanes 2, 6, and 10); 200 nM (lanes 3, 7, and 11); 400 nM (lanes 4, 8, and 12). Panel B, DNA ligase assays performed in panel A were quantified using the Storm PhsophoImager (Amersham Biosciences). Duplex N was incubated with increasing amounts of HU ({square}), with 0.1 unit of DNA ligase and increasing amounts of HU ({circ}), or with 0.01 units of DNA ligase and increasing amounts of HU ({diamondsuit}).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
After exposure to ionizing radiation, the individual damage in each clustered lesion is expected to be repaired via the BER pathway. The processing of DNA damage via the BER pathway generates single strand breaks or small gaps as repair intermediates. These repair intermediates require DNA polymerase and DNA ligase to complete the repair. Therefore, if a nick generated from the initial repair of one of the lesion within the clustered lesion site is not sealed before initiating the repair of additional lesions, a DNA double strand break will be generated. The uncoordinated repair of clustered lesions will lead to an increase in the cellular level of double strand breaks.

In vitro experiments from this laboratory showed that the human DNA-binding protein KU helps to reduce the formation of frank double strand breaks by inhibiting the activity of DNA glycosylase (endonuclease III) in removing the base lesion opposite to a nick (15). Furthermore, KU has a high affinity for DNA ends and will tether the two ends of a double strand break, thus reducing the possibility of misjoining. It is interesting to note that despite the fact that KU inhibits the incision activity of endonuclease III on DNA containing a nick in close proximity opposite to a dihydrouracil (15), KU has little or no inhibitory activity on DNA ligation (28). However, it is not known whether the binding of KU to a nick will lead to any appreciable inhibition of repair synthesis by human polymerase {beta} or other polymerases such as Pol {lambda} and Pol µ that might be involved in the non-homologous rejoining of double strand breaks (3031).

To further understand the role of DNA nick-binding proteins on the repair of closely opposed lesions, we decided to examine the role of the HU protein of E. coli. HU is a basic DNA-binding protein that constitutes one of the 12 capsid proteins of E. coli that bind to E. coli genome (32). In addition to its nonspecific double strand DNA-binding activity, HU protein has a high affinity for various replicative and recombinational DNA structures (2021). HU also exhibits high affinity for DNA nicks and small gaps, and the Kd for binding to nick and small gap is around 2–4 nM (20, 23). DNA nicks and small gaps are intermediates generated during DNA repair via the base excision repair pathway. In this study we showed that similar to KU, increasing concentrations of HU led to an increased inhibition of endonuclease III activity on DHU. Inhibition of endonuclease III activity was only observed when an opposing nick was close to DHU. The nick-dependent inhibition exerted by HU on endonuclease III activity thus suggests that HU plays a novel role in limiting the ability of endonuclease III to produce additional nick within a clustered lesion site before the intermediary nick is fully repaired. It is proposed that the nick-dependent inhibition of DNA glycosylase activity (in this study, endonuclease III activity) by HU will allow DNA polymerase I and DNA ligase to fully repair the nick or gap before endonuclease III or other DNA glycosylases begin to initiate the repair of additional lesions. These data therefore suggest that in a cell, when the repair of one of the base damage within a cluster lesion site is initiated, the generation of a one base gap or nick should lead to substantial increase in the affinity of HU protein for this damage site. Normally HU has a low affinity for double-stranded DNA (Kd = 25,000 nM); however, the affinity of HU for DNA containing a nick is four orders of magnitude higher than a double-stranded DNA with a Kd around 2 to 8 nM (20, 23). HU is involved in several other cellular processes including the formation of the nucleoid structure, DNA replication, and recombination. HU is present in E. coli at high abundance, and it was estimated that E. coli has ~30,000 molecules of HU protein per cell (33). In comparison, there are only about 200 molecules of endonuclease III per cell.2 In this study, we showed that HU binding to a nick did not inhibit the activities of both Pol I and DNA ligase. The binding of HU to a nick thus allows the Pol I and DNA ligase to fully repair the nick while inhibiting the activity of endonuclease III on other lesions that are located in close proximity (Fig. 8). Once the DNA is completely repaired, affinity of HU at the clustered site will decrease substantially, thus leading to the rapid dissociation of HU from the DNA. The dissociation of HU from the damaged site will allow endonuclease III to initiate the repair of the remaining lesion. The sequential repair of closely opposed lesions thus avoids the formation of a potentially lethal and mutagenic double strand break intermediate (Fig. 8).



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FIG. 8.
Proposed role of E. coli HU protein in the repair of closely opposed lesions. Endo III, endonuclease III; Exo III, exonuclease III.

 

We showed that in this study, significant inhibition of endonuclease III activity was observed only when the nick was within two nucleotides from the base lesion. When the distance of the nick was 4 and 6 nucleotides away, inhibition of endonuclease III activity by HU decreased to about 35 and 14%, respectively. Because the inhibition of endonuclease III activity by HU was observed to be similar when the nick is two nucleotides, either 3' or 5' from DHU, it is therefore likely that the binding of HU to the nick is symmetrical. These observations thus suggest that HU heterodimer probably covers about five nucleotides footprint, which is consistent with its small size of only 18 kDa. KU 70/80 complex was shown to have a footprint of about 25 nucleotides (34), and considering the molecular mass of KU, which has a molecular mass of 150 kDa, a footprint of 5 nucleotides for HU is thus reasonable. It has been estimated that damage within a clustered lesion can have a spread of up to 20 nucleotides (7), and at physiological pH, two breaks within a distance of 15–20 nucleotides can lead to double strand breaks. The relatively small footprint of HU when it binds to a nick thus suggests that other proteins might be interacting with HU to generate a larger footprint that can contribute more effective inhibition of the glycosylase activity when the nick is further away from the opposing base damage.

In human cells, double strand breaks are repaired predominantly by nonhomologous end joining activity. In contrast, in bacteria such as E. coli, a double strand break is repaired through homologous recombination. Because bacteria are haploid; the ability to prevent the generation of excessive double strand breaks is crucial for their survival. It is interesting to point out that E. coli mutants lacking HU protein are hyper-sensitive to both UV and ionizing radiation (3637). Because HU is required for homologous recombination, the increased sensitivity of mutants to ionizing radiation could be due to a reduced recombination activity. However, the increased radio-sensitivity could also be due to the result of increased formation of double strand breaks generated from the uncoordinated repair of clustered lesion. The lack of cellular HU protein will lead to a significant increase in the uncoordinated repair of clustered lesions. It is important to point out that E. coli triple mutant cells (fpg nth nei) lacking the major DNA glycosylases, exhibit increased survival toward ionizing radiation as compared with either the single mutants or the wild type cells (35). The increased survival in the triple mutant could be due to a decrease in the ability of the mutant cell to process base lesion within the clustered lesion, thus led to decrease in formation of double strand breaks. However, the increased survival might be accompanied by an increase in mutation frequency, possibly the result of mutagenic bypass of the unrepaired base lesions. Because HU is involved in many cellular processes and is also a constituent of the nucleoid capsid complex, the amount of HU protein available for sequential repair might be rate-limiting. At higher doses of ionizing radiation, significant amounts of HU might be tied up with nicks generated by ionizing radiation, thus leading to a significant reduction of HU that is available for sequential repair of clustered lesions. It is therefore interesting to find out whether overproduction of HU protein in E. coli wild type cells will lead to increased resistance toward ionizing radiation. Furthermore, it is also expected that the increased radioresistance will not be accompanied by a significant increase in the overall mutation frequency.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant CA 90860. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Frontier Collaborative Research Center, Tokyo Institute of Technology, Yokohama 226, 8503, Japan. Back

§ To whom correspondence should be addressed: Division of Cancer Biology, Dept. of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Ave., Atlanta, GA 30303. Tel.: 404-616-6951; Fax: 404-616-5689; E-mail: ykow{at}emory.edu.

1 The abbreviations used are: AP, abasic; BER, base excision repair; DHU, dihydrouracil; Pol I, polymerase I. Back

2 Y. W. Kow, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Bernard Weiss for critical reading of the manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. von Sonntag, C. (1987) The Chemical Basis of Radiation Biology, Taylor and Francis, London
  2. Hall, E. J. (1994) Radiobiology for the Radiobiologist, J. B. Lippincott Company, Philadelphia
  3. Ward, J. F. (1988) Progr. Nucl. Acids Mol. Biol. 35, 95–125
  4. Sutherland, B. M., Bennett, P. V., Sidorkina, O., and Laval, J. (2000) Biochemistry 39, 8026–8031[CrossRef][Medline] [Order article via Infotrieve]
  5. Sutherland, B. M., Bennett, P. V., Sidorkina, O., and Laval, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 103–108[Abstract/Free Full Text]
  6. Chaudhry, M. A., and Weinfeld, M. (1995) J. Mol. Biol. 249, 914–922[CrossRef][Medline] [Order article via Infotrieve]
  7. Ward, J. F. (1981) Radiat. Res. 86, 185–195[Medline] [Order article via Infotrieve]
  8. Hall, E. J. (1993) Radiology for the Radiologist, J. B. Lippincott Company, Philadelphia
  9. Freidberg, E. C., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagenesis, American Society of Microbiology Press, Washington, D. C.
  10. Wilson. D. W., Engelward, B. P., and Samson, L. (1998) in DNA Repair in Prokaryotes and Lower Eukaryotes (Nickoloff, J. A., and Hoekstra, M. F., eds.) pp. 29–64, Humana Press, Totowa, New Jersey
  11. Harrison, L., Hatahet, Z., Purmal, A. A., and Wallace, S. S. (1998) Nucleic Acids Res. 26, 932–941[Abstract/Free Full Text]
  12. David-Cordonnier, M-H., Laval, J., and O'Neill, P. (2000) J. Biol. Chem. 275, 11865–11873[Abstract/Free Full Text]
  13. Takeshita, M., Chang, C. N., Johnson, F., Will, S., and Grollman, A. P. (1987)) J. Biol. Chem. 262, 10171–10179[Abstract/Free Full Text]
  14. Bourdat, A. G., Gasparutto, D., and Cadet, J. (1999) Nucleic Acids Res. 27, 1016–1024
  15. Hashimoto, M., Donald, C. D., Yannone, S. M., Chen, D. J., Roy, R., and Kow, Y. W. (2001) J. Biol. Chem. 276, 12827–12831[Abstract/Free Full Text]
  16. Yao, M., and Kow, Y. W. (1995) J. Biol. Chem. 270, 28609–28616[Abstract/Free Full Text]
  17. Asahara, H., Wistort, P. M., Bank, J. F., Bakerian, R. H., and Cunningham, R. P. (1989) Biochemistry 10, 4444–4449
  18. Yao, M., and Kow, Y. W. (1997) J. Biol. Chem. 272, 30774–30779[Abstract/Free Full Text]
  19. Pellegrini, O., Oberb, J., Pinson, V., and Rouviere-Yaniv, J. (2000) Biochimie (Paris) 82, 1–13[Medline] [Order article via Infotrieve]
  20. Kamashev, D., and Rouviere-Yaniv, J. (2000) EMBO J. 19, 6527–6535[Abstract/Free Full Text]
  21. Kamashev, D., Balandina, A., and Rouviere-Yaniv, J. (1999) EMBO J. 18, 5434–5444[Abstract/Free Full Text]
  22. Bonnefoy, E., Takahashi, M., and Rouviere-Yaniv, J. (1994)) J. Mol. Biol. 242, 116–129[CrossRef][Medline] [Order article via Infotrieve]
  23. Castaing, B., Zelwer, C., Laval, J., and Boiteux, S. (1995) J. Biol. Chem. 270, 10291–10296[Abstract/Free Full Text]
  24. Prasad, R., Lavrik, O. I., Kim, S. J., Kedar, P., Yang, X. P., Vande Berg, B. J., and Wilson, S. H. (2001) J. Biol. Chem. 276, 2411–2414[Abstract/Free Full Text]
  25. Lyamichev, V. D., Brow, M. A., and Dahlberg, J. E. (1993) Science 260, 778–783[Medline] [Order article via Infotrieve]
  26. MacNeill, S. A. (2001) Curr. Biol. 11, R842-R844[CrossRef][Medline] [Order article via Infotrieve]
  27. Yao, M., and Kow, Y. W. (1996) J. Biol. Chem. 271, 30672–30676[Abstract/Free Full Text]
  28. Ramsden, D. A., and Gellert, M. (1998) EMBO J. 17, 609–614[Abstract/Free Full Text]
  29. Hodges-Garcia, Y., Hagerman, P. J., and Pettijohn, D. E. (1989) J. Biol. Chem. 264, 14621–14623[Abstract/Free Full Text]
  30. Ramadan, K., Mega, G., Shevelav, I. V., Villani, G., Blanco, L., and Hubschen, U. (2003) J. Mol. Biol. 328, 63–72[CrossRef][Medline] [Order article via Infotrieve]
  31. NickMcEhinny, S. A., and Ramsden, D. A. (2003) Mol. Cell. Biol. 23, 2309–2315[Abstract/Free Full Text]
  32. Ishihama, A., and Azam, T. A. (1999) J. Biol. Chem. 274, 33105–33113[Abstract/Free Full Text]
  33. Rouviere-Yaniv, J. (1978) Cold Spring Harbor Symp. Quant. Biol. 42, 439–447[Medline] [Order article via Infotrieve]
  34. de Vries, E., van Driel, W., Bergsma, W. G., Arnberg, A. C., and van der Vliet, P. C. (1989) J. Mol. Biol. 208, 65–78[Medline] [Order article via Infotrieve]
  35. Jiang, D., Hatahet, Z., Blaisdell, J. O., Melamede, R. J., and Wallace, S. S. (1997) J. Bacteriol. 197, 3773–3782
  36. Boubrik, F., and Rouviere-Yaniv, J. (1995) Proc. Natl. Acad. Sci. (U. S. A.) 92, 3958–3962[Abstract/Free Full Text]
  37. Li, S., and Waters, R. (1998) J. Bacteriol. 180, 3750–3756[Abstract/Free Full Text]




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