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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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
[
-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).
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RESULTS |
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.
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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.
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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.
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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.
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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
-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 -eliminated
oligodeoxynucleotide; P2, the 5'-end of the
-eliminated oligodeoxynucleotide; =, the Schiff base between
E and S. k2, the rate of base
release; k3, the rate of -elimination.
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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.
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DNA Cleavage by hNth1 Is Stimulated by
His6YB-1--
In order to measure the rate constants of
base release (k2), and
-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.
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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
-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.
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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
EP2) nor the
release of the final product (EP2
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
-elimination product. The concept that
the Schiff base intermediate represented by E=S2
is in steady state equilibrium with the aldehyde and the primary
-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 -eliminated
oligodeoxynucleotide. P2, the 5'-end of the
-eliminated oligodeoxynucleotide. =, the Schiff base between
E and S; k2, the rate of base
release; k3, the rate of -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.
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 |
DISCUSSION |
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
-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
-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
(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
, 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.