Stimulation of Human Endonuclease III by Y Box-binding Protein 1 (DNA-binding Protein B)

INTERACTION BETWEEN A BASE EXCISION REPAIR ENZYME AND A TRANSCRIPTION FACTOR*

Dina R. MarensteinDagger , Maria T. A. OcampoDagger , Michael K. ChanDagger , Alvin Altamirano§, Ashis K. Basu§, Robert J. BoorsteinDagger , Richard P. Cunningham, and George W. TeeborDagger ||

From the Dagger  Department of Pathology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016, the § Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, and  Department of Biological Sciences, The University at Albany, SUNY, Albany, New York 12222

Received for publication, February 20, 2001, and in revised form, April 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human endonuclease III (hNth1) is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by reactive oxygen species, ionizing, and ultraviolet radiation. Using duplex 2'-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth1 was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are also members of the endonuclease III family. This difference in rates contrasts with the equality of rates found in Escherichia coli and Saccharomyces cerevisiae endonuclease III homologs. A yeast two-hybrid screen for potential modulators of hNth1 activity revealed interaction with the damage-inducible transcription factor Y box-binding protein 1 (YB-1), also identified as DNA-binding protein B (DbpB). The in vitro addition of His6YB-1 to hNth1 increased the rate of DNA glycosylase and AP lyase activity. Analysis revealed that YB-1 affects the steady state equilibrium between the covalent hNth1-AP site Schiff base ES intermediate and the noncovalent ES intermediate containing the AP aldehydic sugar and the epsilon -amino group of the hNth1 active site lysine. This equilibrium may be a checkpoint in modulating hNth1 activity.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA excision repair effects the removal of modified and damaged bases and abasic sites formed through the action of endogenous and exogenous genotoxic agents such as ultraviolet and ionizing radiation, electrophilic chemicals, and reactive radical species generated by metabolic oxidative stress. Excision of damaged DNA is accomplished via two major pathways, nucleotide excision repair (NER)1 and base excision repair (BER). NER effects DNA repair via the formation of protein complexes that, in eukaryotic cells, involve over 30 gene products. In contrast, BER has been reconstituted in vitro using far fewer components, and until recently, there has not been evidence of multiprotein complex formation in the initial events of BER (1, 2). However, recent reports have suggested that formation of complexes between BER enzymes and other proteins can stimulate damage recognition or excision (1, 3-5). For example, the NER 3' endonuclease XPG has been reported to stimulate the in vitro activity of the human homologue of the Escherichia coli BER enzyme endonuclease III (hNth1) (1, 3).

hNth1, first identified in our laboratory (6) and subsequently cloned by us and another group (7, 8), is a bifunctional DNA glycosylase/AP lyase whose substrates are virtually identical to those of its bacterial and yeast homologues (e.g. oxidation and hydration products of pyrimidines such as 5-hydroxycytosine, 5-hydroxyuracil, and cytosine and uracil hydrate). However, we and others have observed that the apparent kcat of hNth1 is much lower than that of the bacterial and yeast enzymes (7-9). The stimulatory effect of XPG on hNth1 activity in vitro suggested that in mammalian cells other proteins might interact with hNth1 and modify its catalytic properties.

To identify such proteins, we performed a yeast two-hybrid screen using hNth1 as bait. We identified a specific interaction with the human damage-inducible transcription factor Y box-binding protein 1 (YB-1), also identified as DNA binding protein B (DbpB). The physical and functional interaction of YB-1 (DbpB) with hNth1 was assayed in vitro, where we observed that the interaction of hNth1 with YB-1 resulted in the stimulation of hNth1 catalytic activity. Interestingly, we did not recover XPG as an interacting protein. The effect of YB-1 on hNth1 substrate processing has provided insight into the mechanism of action of hNth1, for which we propose a kinetic model consistent with the data obtained through our analysis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins-- The hNth1 cDNA was cloned into the pGEX6P1 vector (Amersham Pharmacia Biotech). Expression of recombinant hNth1 was induced as described previously (7). The protein was expressed as a glutathione S-transferase fusion protein for purification on a glutathione column. Following purification, the glutathione S-transferase moiety was removed using Prescission Protease (Amersham Pharmacia Biotech), leaving 5 residues remaining on the amino terminus of the recombinant hNth1. hNth1 concentration was quantified using an extinction coefficient of 1 OD at A410 = 69.4 µM for the C-terminal cubane 4[Fe-S] cluster (10).

The full-length cDNA of YB-1 (DbpB) was a gift from Dr. Peter E. Newburger (University of Massachusetts Medical Center). Six histidine codons were added to the YB-1 sequence via polymerase chain reaction, and the resulting fragment was cloned into pBacPAK9 vector (CLONTECH). His6YB-1 was expressed in Trichoplusia ni cells using the BacPAK Baculovirus Expression system from CLONTECH. The virus was produced using the Baculogold Baculovirus kit in SF9 cells. Protein expression was done in 5B cells. The cells were harvested and lysed 2 days postinfection in buffer containing 50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA with 0.1 mM Na3VO4, 10 µg/ml tosylphenylalanyl chloromethyl ketone, 10 µg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, 1 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. His6YB-1 was bound to a Ni2+-NTA-agarose column (Qiagen) by gravity flow and washed with buffer containing 50 mM sodium phosphate, pH 6.2, 300 mM NaCl, 10% glycerol, 1% Tween, and 5 mM imidazole. The protein was eluted by applying an imidazole gradient in wash buffer without Tween, followed by concentration and dialysis to remove imidazole using Microcon centrifuge concentrating filters (Millipore Corp.). His6YB-1 was visualized by SDS-PAGE and Coomassie Blue staining and quantitated by densitometry against glutathione S-transferase as a standard using the Molecular Imager FX System with Quantity One Software (Bio-Rad).

2'-Deoxyribose Oligonucleotides-- 2'-Deoxyribose oligonucleotides were synthesized by the NYU School of Medicine Department of Cell Biology. 2'-Deoxyribose oligonucleotides were deblocked, deprotected, and purified by 20% denaturing PAGE. The AP site-containing substrate was generated from a 26-mer uracil-containing 2'-deoxyribose oligonucleotide of the sequence d(CGCGAAACGCCTAGUGATTGGTAGGG). The 5-hydroxy-2'-deoxyuracil (5-ohUra)-containing 2'-deoxyribose oligonucleotide was purchased from Oligos Etc. with the same sequence as above with the 5-ohUra residue at position 15 in place of uracil. A 30-mer 2'-deoxyribose oligonucleotide substrate containing a thymine glycol (Tg) residue at position 13 d(GATCCTCTAGCGTgCGACCTGCAGGCATGCA) was prepared as follows; a 9-mer d(AGAGTgCGAC) was prepared by KMnO4 oxidation as described by McNulty et al. (11). Part of the purified 9-mer was enzymatically digested to component nucleosides and analyzed by reverse phase HPLC. Chromatographic analysis at 254 nm showed the anticipated amounts of dA, dG, and dC, but no detectable dT from this oligonucleotide. However, the Tg-containing 2'-deoxyribose nucleoside could be detected at 220 nm. Additional evidence for the presence of Tg in the 9-mer was obtained by mass spectral analysis by electrospray ionization. Analysis of the Tg-containing 9-mer gave a monoisotopic mass of 2780.4, which was in excellent agreement with the theoretical monoisotopic mass of 2780.5. The Tg 9-mer was 5'-end-phosphorylated and ligated to an 8-mer on its 5'-end and a 5'-end phosphorylated 13-mer on its 3'-end in the presence of a complementary 25-mer oligonucleotide. The ligated 30-mer, d(GATCCTCTAGAGTgCGACCTGCAGGCATGCA), in which the Tg was located at position 13 was purified by PAGE and desalted.

2'-Deoxyribose oligonucleotides were labeled at the 5'-end using [gamma -32P]ATP (PerkinElmer Life Sciences) and T4 polynucleotide kinase (Life Technologies, Inc.) and then annealed to the corresponding complementary strand in a 1:2 ratio. All complementary strands contained an adenine residue opposite the AP site, Tg residue, or 5-ohUra residue. To obtain a duplex 2'-deoxyribose oligonucleotide containing an AP site opposite A, the duplex with the U:A pair was treated with 0.3 units/µl of uracil DNA glycosylase (Life Technologies).

Yeast Two-hybrid Screen-- Full-length hNth1 was cloned into pEG202 and transformed into Saccharomyces cerevisiae strain EGY48, with six lexA operators directing transcription from a LEU2 reporter gene, along with the reporter plasmid pSH18-34, with 8 lexA operators directing transcription of the lacZ gene. A Jurkat cDNA library in the expression plasmid pJG45 was a gift of Dr. X. H. Sun (NYU School of Medicine, Department of Cell Biology) and was screened for growth on selective medium and LacZ expression in the presence of the bait protein. Yeast strain EGY48 and all associated plasmids for the yeast two-hybrid screen were a gift from Dr. H. L. Klein (NYU School of Medicine, Department of Biochemistry).

Pull-down Assay-- HeLa cell extract was prepared by lysing 3 × 107 HeLa cells in 1 mM dithiothreitol, 50 mM NaCl, 50 mM HEPES, pH 7.8, 10 mM EDTA, and mammalian protease inhibitor mixture (Sigma). A cell suspension was sonicated on ice and centrifuged at 4 °C at 20,000 × g for 15 min. The supernatant was collected, and the protein content was quantitated via the Bradford assay (Bio-Rad). Purified His6YB-1 protein expressed in insect cells was purified as above and then rebound to Ni2+-NTA beads overnight, washed, and incubated with 1100 µg of HeLa cell extract for 20 min at 4 °C in the presence of 20 mM imidazole. The beads were then washed in buffer containing 20 mM imidazole. Boiling the beads in Laemmli sample buffer followed by separation by SDS-PAGE isolated the bound proteins. Following a Western transfer, an immunoblot was performed for the identification of hNth1 using a rabbit polyclonal anti-hNth1 antibody produced for our laboratory by HRP Inc. (Denver, PA).

DNA Glycosylase Assay-- Poly(dA-[3H]dT)·poly(dA-[3H]dT) (~1200 base pairs) containing 8-12% Tg residues and poly(dG-[3H]dC)·poly(dG-[3H]dC) (~750 base pairs) containing 8-12% cytosine hydrate residues were made as previously described (7). The content of modified base was measured by performing limit digests of the substrates with E. coli endonuclease III (Nth). Assay mixtures contained 50 mM HEPES, pH 7.6, 100 mM KCl, 0.1 mg/ml bovine serum albumin, 1 mM EDTA, 0.1 mM dithiothreitol, in which 1 pmol of hNth1 was incubated with 100 ng of substrate without or with His6YB-1, at 37 °C for 1 h in a final volume of 50 µl. The assays were stopped by the addition of 100 µl of bovine serum albumin (50 mg/ml) and 1 ml of HPLC grade acetone. The tubes were kept at -20 °C for 20 min to precipitate the DNA and proteins. The assay tubes were then centrifuged at 8000 × g for 15 min, after which the supernatant was transferred to a new tube, and the acetone evaporated. The residue was resuspended in 300 µl of water and analyzed by liquid scintillation counting.

Cleavage Assays-- Assays were performed at 37 °C in buffer containing 50 mM HEPES, pH 7.6, 100 mM KCl, 0.1 mg/ml bovine serum albumin, 1 mM EDTA, and 0.1 mM dithiothreitol. Enzyme, protein, and substrate were diluted to working conditions in assay buffer and equilibrated at 37 °C. Single turnover assays contained 30 nM 32P-5'-end-labeled 2'-deoxyribose oligonucleotide duplex and 300 nM hNth1 with or without 300 nM His6YB-1 in a volume of 120 µl. His6YB-1 and hNth1 were incubated together for 2 min prior to initiation of the assay upon the addition of substrate. To measure strand cleavage, 10-µl aliquots were removed at the indicated time periods and treated with an equal volume of loading dye (95% deionized formamide, 10 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). To measure base release, 10-µl aliquots were removed at the indicated time periods and treated with 5 µl of 0.5 M putrescine, pH 8.0 (12). The treated assay mixtures were then heated at 95 °C for 5 min, followed by the addition of 15 µl of loading dye. Multiple turnover assays were performed individually in 20-µl volumes, containing reaction buffer, 100 nM 2'-deoxyribose oligonucleotide substrate duplex, and 10 nM hNth1 with or without 10 nM His6YB-1. The assays were stopped by snap freezing in ethanol and dry ice and treated as described above. Samples were then heated at 95 °C for 5 min, and products were separated by 20% PAGE in 7 M urea and TBE. Products were analyzed quantitatively via phosphorimaging using a Molecular Imager FX System with Quantity One Software (Bio-Rad). Data was weighted using GraFit 4.03 software (Erithacus Software).

Cross-linking of Enzyme to 2'-Deoxyribose Oligonucleotide Substrate (Enzyme Trapping)-- Assay conditions were the same as for multiple turnover assays except that assay mixtures were treated for 2 min with 5 µl of 0.5 M NaBH4 following incubation (12). After the addition of Laemmli SDS-loading buffer, reaction products were separated by 12% SDS-PAGE and analyzed as above.

Nicking Assay-- Supercoiled pBR322 (Promega) DNA containing AP sites was prepared by heating at 70 °C for 10 min in 50 mM sodium acetate, pH 4.5, 50 mM NaCl. The solution was then neutralized by the addition of HEPES, pH 8.0, and EDTA to yield a final concentration of 10 mM HEPES and 50 µM EDTA. Limit digestion with E. coli Nth yielded almost 100% form II DNA, indicating that the distribution of induced AP sites was at least 20 sites/molecule of plasmid DNA. Enzyme nicking assays were performed in the HEPES reaction buffer listed above in a volume of 10 µl, containing 500-750 ng of treated pBR322 DNA and 5 nM hNth1 with or without 10 nM His6YB-1. The assays were incubated at 37 °C for 30 min, terminated by the addition of 6× Loading Dye (Promega), and separated on a 0.7% agarose gel. The bands were visualized by ethidium bromide staining and quantitated by densitometry using the Molecular Imager FX System with Quantity One Software (Bio-Rad).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

YB-1 Interacts with hNth1 in a Yeast Two-hybrid Screen-- A yeast two-hybrid screen was performed in which the full-length hNth1, originally cloned in our laboratory (7), was used as a bait protein fused to the LexA DNA-binding protein (pEG202-hNth1). A Jurkat cDNA library fused to the pJG45 GAL4 transcriptional activation domain was used in a screen of 4 × 107 colony-forming units. An initial screen for growth on selective medium and LacZ expression yielded 140 positive colonies. Of 27 rescued cDNAs sequenced, the sequences of 25 matched the YB-1/EFIA family as revealed by a BLAST search of the nonredundant GenBankTM + EMBL + DDBJ + PDB data base (13). Several longer cDNAs were fully sequenced and found to have 97% nucleotide sequence identity with the canonical YB-1 sequence (14).

hNth1 Interacts with His6YB-1 in Vitro-- To determine whether interaction between YB-1 and hNth1 occurred in vitro, purified His6YB-1 was rebound to Ni2+-NTA-agarose beads, and HeLa cell extract containing endogenous hNth1 was applied to the column. As shown in Fig. 1, hNth1 was retained on the His6YB-1 column but not on the control column.


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Fig. 1.   Specific interaction between hNth1 and His6YB-1 proteins in vitro. 1100 µg of HeLa extract was applied to Ni2+-NTA resin with bound His6YB-1 and to a control column of Ni2+-NTA resin. After washing, bound proteins were separated by SDS-PAGE and analyzed by Western blot with polyclonal anti-hNth1 antibody. 110 µg of HeLa extract were loaded into the HeLa input lane.

His6YB-1 Stimulates hNth1 Activity against High Molecular Weight Substrates Containing AP Sites, Tg, or Cytosine Hydrate Residues-- To determine whether there was an effect of YB-1 on hNth1-mediated catalysis, the activity of a fixed amount of hNth1 was assayed in the presence of varying concentrations of YB-1 using three different high molecular weight substrates: supercoiled plasmid DNA containing AP sites created by acid hydrolysis of purines; poly(dA-[3H]dT)·poly(dA-[3H]dT) containing multiple Tg residues formed by oxidation with osmium tetroxide; and poly(dG-[3H]dC)·poly(dG-[3H]dC) containing multiple cytosine hydrate residues formed by ultraviolet irradiation. Table I summarizes the effect of YB-1 on hNth1 AP lyase activity as determined by a nicking assay using depurinated supercoiled plasmid DNA containing AP sites as substrate. The nicking activity of hNth1 was stimulated more than 2-fold. The presence of near equimolar amounts of YB-1 significantly increased hNth1-catalyzed release of cytosine hydrate from UV-treated poly(dG-[3H]dC)·poly(dG-[3H]dC) and the release of Tg from osmium tetroxide-treated poly(dA-[3H]dT)·poly(dA-[3H]dT). YB-1 alone had no activity against these substrates, and the addition of equimolar and excess amounts of bovine serum albumin as a protein control had little effect on hNth1 activity. Since YB-1 has a theoretical pI of 9.9, the reactions were also carried out in the presence of equimolar and excess amounts of spermidine, as a control for nonspecific effects of basic proteins. The addition of spermidine also had no effect on hNth1 activity.

                              
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Table I
His6YB-1 stimulation of hNth1 activity against a variety of DNA substrates
Assays were performed as described under "Experimental Procedures." Values are mean and S.E. of at least two experiments.

His6YB-1 Increases hNth1 Processing of 2'-Deoxyribose Oligonucleotide Substrates-- The kinetics of enzyme-catalyzed nicking of 2'-deoxyribose oligonucleotide substrates containing a single lesion were investigated using substrates containing either a single AP site, a single Tg residue, or a single 5-ohUra residue. The presence of YB-1 increased the amount of enzyme-mediated cleavage of all three substrates as shown in Figs. 2, 3, and 7. The effect of YB-1 could be seen at as little as a 1:4 molar ratio of YB-1 to hNth1, as shown in Fig. 2. In cleavage assays using duplex 2'-deoxyribose oligonucleotide substrates, hNth1 activity was lower than that of E. coli Nth by nearly 2 orders of magnitude (Ref. 7 and data not shown). However, in the DNA glycosylase assays measuring base release from high molecular weight substrates, the difference was only 2-4-fold (data not shown). These results suggested that there might be a difference in the relative rates of DNA glycosylase and AP lyase activities of hNth1.


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Fig. 2.   Effect of increasing concentrations of His6YB-1 on DNA glycosylase and AP lyase activities of hNth1. A, phosphor image of separated cleavage assay products using AP site-containing 2'-deoxyribose oligonucleotide duplex substrate. 33 nM (660 fmol) of AP site-containing 32P 5'-end labeled duplex 2'-deoxyribose oligonucleotide was incubated with 20 nM (400 fmol) hNth1 in the presence of increasing concentrations of His6YB-1 for 5 min. Reactions were stopped by snap freezing prior to the addition of formamide loading dye. The last lane shows the cleavage of AP sites by putrescine alone. B, plotted data of cleavage assay using 5-ohUra containing duplex 2'-deoxyribose oligonucleotide substrate. 11 nM (220 fmol) 5-ohUra containing 32P-5'-end-labeled duplex 2'-deoxyribose oligonucleotide was incubated with 20 nM (400 fmol) hNth1 in the presence of increasing concentrations of His6YB-1 for 2.5 min. Duplicate reactions were stopped by snap freezing, after which one set was treated with putrescine (gray bars). Products were separated by denaturing gel electrophoresis and analyzed by phosphorimaging.

The Rate of the DNA Glycosylase Activity of hNth1 Is Much Greater than That of Its AP Lyase Activity-- The catalysis of DNA strand cleavage by DNA glycosylase/AP lyase enzymes has long been thought to occur concomitantly with base removal (15). However, a recent analysis of the mechanism of action of a member of the endonuclease III family, murine 8-oxoguanine-DNA glycosylase (mOgg1), revealed that the rate of the AP lyase cleavage reaction was much slower than that of the DNA glycosylase activity at physiological pH (12). Given the results described in the previous paragraph and the data reported for mOgg1, we investigated whether the AP lyase activity of hNth1 was concurrent with its DNA glycosylase activity.

Quantitative measurement of AP sites formed via the DNA glycosylase activity of hNth1 was accomplished by treating the assay mixture with putrescine, resulting in the cleavage of existing AP sites (see last lane in Fig. 2A), while having no effect on 2'-deoxyribose oligonucleotide substrates with the N-glycosylic bond between the modified base and the sugar phosphate backbone still intact (12). Thus, the cleavage of the DNA substrate observed in the absence of putrescine treatment represents the true AP lyase activity of the enzyme. The quantitative difference in cleaved product between the putrescine and non-putrescine-treated reactions represents the number of AP sites generated by the DNA glycosylase activity of the enzyme that have not been enzymatically cleaved via AP lyase activity.

In assays using 5-ohUra (data not shown) and Tg-containing duplex 2'-deoxyribose oligonucleotide substrates, the initial rate of release of the modified base by the enzyme was much greater than the rate of DNA strand cleavage (Fig. 3).


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Fig. 3.   Effect of His6YB-1 on kinetics of hNth1 reaction with a Tg-containing 2'-deoxyribose oligonucleotide duplex. 100 nM (2 pmol) of TG-containing 32P 5'-end labeled duplex 2'-deoxyribose oligonucleotide was incubated with 10 nM (200 fmol) hNth1 at 37 °C with (squares) and without (circles) 10 nM (200 fmol) His6YB-1 in a 20-µl volume. Duplicate reactions were stopped by snap freezing, after which one set was treated with putrescine (open symbols), prior to separation and analysis.

His6YB-1 Increases both hNth1-catalyzed Base Release and DNA Cleavage-- As shown in Fig. 3, not only did YB-1 stimulate hNth1-mediated cleavage of the substrate, but the presence of YB-1 also increased hNth1 DNA glycosylase activity, resulting in the generation of more putrescine labile sites by the enzyme. These results suggested that YB-1 affected the overall rate of enzyme catalysis.

Analysis of the hNth1 Reaction Mechanism-- The simplest mechanism consistent with the kinetic data for the activity of hNth1 in the absence of YB-1 is presented in Fig. 4. This scheme illustrates the basic steps of the enzymatic cycle: binding of the substrate (k1, k-1), Schiff base formation effecting base release (DNA glycosylase activity, k2), strand cleavage (AP lyase activity, k3), hydrolysis of the Schiff base (k4, k-4), and product release (k5, k-5). Bifunctional DNA glycosylases/AP lyases catalyze release of modified bases via displacement from the DNA backbone by formation of a transient Schiff base intermediate (E=S2) in which the DNA is covalently linked to the enzyme (16). The Schiff base covalent DNA-enzyme intermediate is resolved via enzyme mediated beta -elimination (k3), which results in strand cleavage and dissociation of the enzyme from the cleaved DNA strand by Schiff base hydrolysis. The rationale behind our experimental approach was to measure each rate constant separately to determine which one was affected by YB-1.


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Fig. 4.   Initial model for hNth1 mechanisms. S1, the substrate with modified base (i.e. Tg or 5-ohUra) intact; S2, the substrate containing an AP site after base release; P1, the 3'-end of the beta -eliminated oligodeoxynucleotide; P2, the 5'-end of the beta -eliminated oligodeoxynucleotide; =, the Schiff base between E and S. k2, the rate of base release; k3, the rate of beta -elimination.

hNth1 Exhibits Non-Michaelis-Menten Kinetics-- In our analysis of hNth1 activity, the enzyme could not be shown to follow Michaelis-Menten pseudo-zero order kinetics with any substrate, including an AP site-containing substrate (Fig. 5). Similar to results reported for the mOgg1 enzyme, product formation did not increase linearly as a function of enzyme concentration (12). Instead, the apparent kcat increased with increasing hNth1 concentration, suggesting positive cooperativity in hNth1 substrate processing. Because of this apparent phenomenon, meaningful Km values for the duplex 2'-deoxyribose oligonucleotide substrates could not be obtained.


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Fig. 5.   v versus Et for hNth1 processing of an AP site containing 2'-deoxyribose oligonucleotide duplex. Increasing concentrations of hNth1 were incubated with an excess (>= 2 µM) of AP site-containing 32P-5'-end-labeled duplex 2'-deoxyribose oligonucleotide substrate. Reactions were stopped by snap freezing prior to the addition of formamide loading dye. Products were separated by denaturing gel electrophoresis and analyzed by phosphorimaging. Values are mean and S.E. of at least two experiments.

DNA Cleavage by hNth1 Is Stimulated by His6YB-1-- In order to measure the rate constants of base release (k2), and beta -elimination (k3), we investigated the reaction under single turnover conditions where [E] [S] (18). Under these conditions all substrate molecules are bound by enzyme. Thus, the rates determining product formation under single turnover conditions are dependent only on k2 and k3. If YB-1 does not affect either k2 or k3, then there should be no stimulation under conditions of single turnover. Fig. 6 illustrates that the stimulatory effect was seen under conditions where neither enzyme turnover nor substrate binding contributed significantly to the rate of the reaction. An increase in AP lyase-mediated AP site cleavage was seen with both the AP site (Fig. 6A) and the Tg residue-containing substrate (Fig. 6B).


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Fig. 6.   Effect of His6YB-1 on single-turnover kinetics of hNth1 with 2'-deoxyribose oligonucleotide duplexes containing an AP site (A) or Tg residue (B). 30 nM 32P-5'-end-labeled duplex 2'-deoxyribose oligonucleotide substrate containing an AP site (A) or Tg residue (B) and 300 nM hNth1 were incubated at 37 °C with (squares) and without (circles) 300 nM His6YB-1 as described under "Experimental Procedures." 10-µl aliquots were taken from the reaction mixture at the indicated times and either quenched with formamide loading buffer (solid squares and circles) or treated with putrescine (open squares and circles) prior to analysis by gel electrophoresis. Products were separated by denaturing gel electrophoresis and analyzed by phosphorimaging. Calculated rate constants are summarized in C.

The release of the modified base by the enzyme occurred rapidly and was not affected by the presence of YB-1, as reflected in the calculated k2 values for the DNA glycosylase reaction in Fig. 6C. In the single turnover assay with the Tg residue-containing substrate, the binding to the substrate and base release appeared to be rapid, while the cleavage of the resulting AP site was clearly the rate-limiting reaction. The data were analyzed using the first order rate equation [P] = A0(1 - exp(-kobst)), where A0 represents the amplitude of the exponential phase and kobs is the rate constant correlated with the reaction. As tabulated in Fig. 6C, YB-1 increased k3 for the Tg-containing substrate by a factor of 4 and by more than a factor of 2 for the AP site containing 2'-deoxyribose oligonucleotide. The differences in k3 values for the two substrates may be due to a difference in their sequence and length. It is possible that YB-1 may also affect k2. However, the DNA glycosylase reaction of hNth1 with the Tg-containing substrate, as well as with a 5-ohUra-containing substrate (data not shown) under these conditions was too rapid to quantitate, and attempts to measure k2 by decreasing reaction temperature to as low as 4 °C were unsuccessful (data not shown).

His6YB-1 Increases Schiff Base Enzyme-Substrate Intermediates-- The data suggesting that YB-1 actually stimulated the beta -elimination-mediated cleavage step without detectably affecting DNA glycosylase activity under single turnover conditions were surprising. We therefore investigated the effect of YB-1 on the steady state concentration of the Schiff base reaction intermediate by tracking the formation of the E=S2 covalent complex over time (Fig. 7). The Schiff base covalent DNA-enzyme intermediate can be trapped via reduction to a stable amine by the action of reducing agents such as NaBH4 or NaCNBH3 (12, 19). Under standard conditions for the assay of hNth1 activity, the addition of YB-1 increased the overall number of trapped enzyme-substrate complexes by a factor of 4 (Fig. 7A). Accordingly, total reaction product was increased by the same amount (Fig. 7B). YB-1 did not cross-link to the DNA on its own (data not shown). Following the kinetic mechanism outlined in Fig. 4, the increase in E=S2 complexes suggests an effect on k2, yet the single turnover data suggest the opposite. The E=S2 and E=P2 complexes can be resolved by SDS-PAGE and are clearly distinguishable in this assay; however, E=P2 was barely detectable under turnover conditions (data not shown), suggesting that k4 is not rate-limiting.


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Fig. 7.   Effect of His6YB-1 on processing of Tg-containing duplex 2'-deoxyribose oligonucleotide substrate. 100 nM Tg-containing 32P-5'-end labeled duplex 2'-deoxyribose oligonucleotide substrate was incubated with 10 nM hNth1 at 37 °C with (squares) and without (circles) 10 nM His6YB-1. Aliquots were taken from the reaction mixture at the indicated times and treated with NaBH4. Products were separated by SDS-PAGE and analyzed by phosphorimaging. A, formation of the enzyme-DNA Schiff base intermediate (E=S2) under turnover conditions as a function of time. B, total substrate processed, the sum of the enzyme-DNA Schiff base intermediate and cleaved product (E=S2 + P2), under turnover conditions.

The fact that YB-1 increased the reaction product of k2 without changing the reaction rate suggested that the model shown in Fig. 4 was not sufficient to accurately describe hNth1 substrate processing. The model of hNth1 activity, shown in Fig. 8, introduces four additional rate constants defining the steady state distribution of the enzyme-substrate intermediate. The model assumes that neither the hydrolysis of the Schiff base enzyme-product intermediate (E=P2 right-arrow EP2) nor the release of the final product (EP2 right-arrow E + P2) is rate-limiting in the overall DNA glycosylase/AP lyase reaction sequence. This model can explain the action of YB-1 on hNth1 lyase activity by suggesting that it drives the steady state equilibrium toward the enzyme-DNA Schiff base intermediate (k6, k-6). This results in an increase in [E=S2] intermediate and therefore an increase in the beta -elimination product. The concept that the Schiff base intermediate represented by E=S2 is in steady state equilibrium with the aldehyde and the primary epsilon -amine of the active site, lysine 212, has been proposed by others (9, 15). We suggest that this equilibrium between the enzyme-substrate Schiff base intermediate and enzyme noncovalently bound to the AP site-containing DNA is a point of regulation of hNth1 activity and is affected by YB-1.


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Fig. 8.   Full kinetic scheme deduced from mechanistic considerations. S1, the substrate with modified base (i.e. Tg or 5-ohUra) intact. S2, the substrate containing an AP site after base release; P1, the 3'-end of the beta -eliminated oligodeoxynucleotide. P2, the 5'-end of the beta -eliminated oligodeoxynucleotide. =, the Schiff base between E and S; k2, the rate of base release; k3, the rate of beta -elimination; k-6 and k6, the steady state of Schiff base resolution; k-7 and k7, the binding and release of the noncovalently bound AP site moiety.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our investigation of the mechanism by which YB-1 stimulates hNth1 activity has provided novel insight into the mechanism of hNth1 activity. Complete analyses of the mechanism of activity of hNth1 have not been reported, and previously published characterizations of the enzyme's activity utilized a variety of nonstandardized assays (7-9, 20). Notably, many assays of hNth1 enzymatic activity have used Tris as buffer (8, 9, 21). We found that Tris stimulated both Nth and hNth1 AP lyase activity in a Tris concentration-dependent manner, whereas buffers such as HEPES or sodium phosphate did not (data not shown).

Although earlier studies identified hNth1 as a DNA glycosylase/AP lyase that effected base excision and beta -elimination reactions via a Schiff base intermediate similar to Nth, the coordination of the two reactions was never documented (7-9, 20). To the best of our knowledge, this is the first report that the DNA glycosylase reactions and the AP lyase reaction of hNth1 are not concurrent and that the rate of AP lyase-mediated strand cleavage is much slower than the rate of DNA glycosylase-mediated base release. These results are similar to the nonconcurrence of base release and strand cleavage reactions reported for mOgg1 at physiological pH (12). However, at pH 6.5, the AP lyase activity was comparable with the DNA glycosylase activity of mOgg1. Interestingly, similar data have been presented for T4 endonuclease V, another DNA repair enzyme that also catalyzes the cleavage of the 3'-phosphate of an AP site by beta -elimination (22). Its pH optimum for AP site cleavage was found to be 5.0, whereas at physiological pH its activity was 4-fold lower (22). In addition, the slow enzyme turnover rates of mOgg1 mirror the reports on the rates of many mammalian BER DNA glycosylases, being much slower than their bacterial or yeast counterparts (20, 21). This has led to the search for interacting or regulatory proteins, which may stimulate the activity of the mammalian BER glycosylases to approximate the rates of their bacterial counterparts in vitro.

We do not yet understand the reason for the apparent increase in Kcat accompanying increases in hNth1 concentration under conditions where [S] [E]. One explanation might be that homodimerization of hNth1 facilitates enzyme function, but we have no positive evidence in that regard. How this apparent cooperativity is manifest and/or advantageous under in vivo conditions awaits further elucidation.

It has been reported that the rate-limiting step of mammalian abasic site BER involving monofunctional DNA glycosylases is the removal of the deoxyribose phosphate moiety by DNA polymerase beta  (23). Studies with mOgg1 and our studies with hNth1 suggest that AP site processing is the rate-limiting step in BER initiated by bifunctional DNA glycosylases/AP lyases. In light of the apparently inefficient processing of AP sites by hNth1 and related enzymes in vitro, what is the in vivo significance of AP lyase activity? Severe oxidative stress or UV irradiation would cause the instantaneous formation of numerous modified bases throughout the genome. In this case, BER via concerted DNA glycosylase/AP lyase activity by bifunctional DNA glycosylases/AP lyases would result in the formation of multiple DNA strand breaks. The delayed AP lyase activity of the enzyme may safeguard the AP site until other components required for the subsequent steps of BER are recruited. In support of this theory, Lindahl and colleagues (1) reported "faint apparent" stimulation of hNth1 AP lyase activity by the BER proteins x-ray repair cross-complementing protein 1 and DNA ligase III. In a similar investigation, Mitra and colleagues (24) recently reported that one of the BER components, apurinic/apyrimidinic endonuclease 1, stimulated the glycosylase activity of human Ogg1 and inhibited its AP lyase activity. This may represent the channeling of AP site processing into specific BER pathways, yet it is unclear why apurinic/apyrimidinic endonuclease 1 catalyzed strand cleavage would be more advantageous than human Ogg1-catalyzed cleavage. However, apurinic/apyrimidinic endonuclease 1 also demonstrated product inhibition with the AP site substrate, suggesting that there may be additional components involved (24).

The relationship between YB-1 and hNth1 may be representative of a mechanism for the coordination of BER with other cellular processes, such as transcription. The link between transcription and DNA repair has been seen mainly with the NER pathway (25), which shares factors such as transcription factor IIH with the transcription complex. There are, however, several examples of BER links to transcription. Apurinic/apyrimidinic endonuclease 1 (REF1/HAP1/APEX1), the human AP endonuclease, has also been identified as a member of the hemin response element-binding protein transcription factor complex and a regulator of several major transcription factors in addition to its role in BER of AP sites (26). Interestingly, the endonuclease activity of apurinic/apyrimidinic endonuclease 1 was found to be stimulated by HSP70, a protein chaperone induced by a variety of stresses (27). Another correlation of BER and transcription involves the stimulation of response element binding of retinoid receptors (retinoic acid receptor and retinoid X receptor) by the T:G mismatch-specific thymine-DNA glycosylase (28). The stimulation of hNth1 by the NER endonuclease XPG may reflect a role for hNth1 in transcription-coupled repair rather than simply an example of shared factors between the two pathways (1, 3).

YB-1 is a multifunctional protein belonging to a family of nucleic acid-binding proteins that are conserved from E. coli to humans (29). YB-1 was originally isolated and characterized based on its DNA binding properties but appears to have a variety of functions, including mRNA selenocysteine insertion sequence binding (14, 30-32). In general, the members of this family contain a central nucleic acid binding domain, also called the cold shock domain, which appears to be conserved from proteins involved in the cold shock response of E. coli (33). These proteins recognize specific DNA sequences, such as an inverted CCAAT repeat termed a "Y box," which regulate the transcriptional activity of specific promoters (30). A number of cell growth-associated genes contain the Y box sequences in their cis-regulatory elements, including proliferating cell nuclear antigen, DNA polymerase alpha , thymidine kinase, epidermal growth factor receptor, multidrug resistance 1, and mammalian major histocompatibility complex class II genes (14, 29, 30, 34).

Accordingly, the expression of YB-1 was up-regulated in cisplatin and multidrug-resistant tumor cell lines, while knockout of YB-1 via transfection of antisense mRNA resulted in increased sensitivity of human cells to the cytotoxic effect of cisplatin, mitomycin C, and UV radiation as compared with drug-sensitive parental lines (35, 36). YB-1 has been reported to undergo translocation from the cytoplasm to the nucleus upon exposure of cells to UV radiation or anticancer agents, suggesting a stress-inducible response, and has also been shown to exhibit preferential binding to DNA containing structural alterations such as cisplatin (37, 38). YB-1 has also been shown to physically interact with several nuclear proteins, including proliferating cell nuclear antigen, which is involved in both NER and BER (2, 39, 40). The preferential binding to structurally altered DNA, its stress-inducible nuclear localization, and its ubiquitous distribution within all tissues of higher organisms suggest that YB-1 may play a role in DNA repair (38).

Having established a physical and functional interaction between a DNA repair enzyme and a pluripotent transcription factor, we are in the process of identifying the regions of hNth1 and YB-1 that effect their interaction in order to understand how this interaction affects the formation of a Schiff base intermediate between enzyme and AP site (E=S2, k-6). To investigate the biological significance of their interaction, we will determine whether there is in vivo colocalization in the nucleus of hNth1 and YB-1 in response to genotoxic stresses. Is the interaction between BER and the Y box family of transcription factors limited to hNth1 and YB-1, or is there a more global relationship between these two pathways? We performed a yeast two-hybrid screen using YB-1 as bait. We recovered hNth1 and nucleolin, corroborating an earlier report (41), as well as YB-1 itself (data not shown), supporting speculation that YB-1 can form multimers (30). Although we identified no other DNA glycosylases via the yeast two-hybrid screen, it is of obvious interest to determine whether YB-1 has any effect on human Ogg1, which is a member of the endonuclease III family and, similarly to hNth1, exhibits disassociation between its DNA glycosylase and AP lyase activities (12). In vitro measurements of BER using hNth1, YB-1, and XPG, together with the downstream effectors of repair will permit assessment of the relative contributions of each protein-protein interaction to the overall rate of BER.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA 16669 and CA 49869 (to G. W. T.), CA 16087 (to Kaplan Cancer Center), 5T32 CA-09161 (to D. R. M.), and NIES-ES 09127 (A. K. B).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Pathology, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-5473; Fax: 212-263-8211; E-mail: george.teebor@med.nyu.edu.

Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101594200

    ABBREVIATIONS

The abbreviations used are: NER, nucleotide excision repair; BER, base excision repair; Nth, E. coli endonuclease III; hNth1, human endonuclease III homologue 1; AP, apurinic/apyrimidinic; Ogg1, 8-oxoguanine-DNA glycosylase; YB-1, Y box-binding protein 1; DbpB, DNA-binding protein B; XPG, xeroderma pigmentosum group G protein; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Tg, thymine glycol; 5-ohUra, 5-hydroxyuracil; HSP70, heat shock protein 70; NTA, nitrilotriacetic acid.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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