substrate specificity of human endonuclease iii (hnth1): effect of

Substrate Specificity of Human Endonuclease III (hNth1): Effect of Human AP

Endonuclease 1 (APE1) on hNth1 Activity

Dina R. Marenstein‡, Michael K. Chan‡, Alvin Altamirano*, Ashis K. Basu*, Robert J.

Boorstein‡, Richard P. Cunningham§ and George W. Teebor‡¶.

‡ Department of Pathology and Kaplan Comprehensive Cancer Center, New York University

School of Medicine, New York, NY 10016. *Department of Chemistry, University of

Connecticut, Storrs, CT 06269. § Department of Biological Sciences, The University at Albany,

SUNY, Albany, NY 12222.

Running Title: Substrate-specific modulation of hNth1 activity by APE1

¶ To whom correspondence should be addressed:

Dept. of Pathology, New York University Medical Center. 550 First Ave., New York, N.Y.

10016. Tel.: 212-263-5473; Fax: 212-263-8211; Email: [email protected].

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SUMMARY

Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-

glycosylase/AP lyase, hNth1, the homolog of E. coli endonuclease III (Nth). In contrast to Nth,

the DNA N-glycosylase activity of hNth1 was 7 fold greater than its AP lyase activity when the

DNA substrate contained a thymine glycol (Tg) opposite adenine (Tg:A) (1). When Tg was

opposite guanine (Tg:G), the 2 activities were of the same specific activity as the AP lyase

activity of hNth1 against Tg:A (2). We now demonstrate that hNth1 was inhibited by the product

of its DNA N-glycosylase activity directed against Tg:G, the AP:G site. In contrast, hNth1 was

not as inhibited by the AP:A site arising from release of Tg from Tg:A. Addition of human AP

endonuclease (APE1) increased dissociation of hNth1 from the DNA N-glycosylase generated

AP:A site, resulting in abrogation of AP lyase activity and an increase in turnover of the DNA

N-glycosylase activity of hNth1. Addition of APE1 did not abrogate hNth1 AP lyase activity

against Tg:G. The stimulatory protein YB-1, (1), added to APE1, resulted in an additive increase

in both activities of hNth1 regardless of base pairing. Tg:A is formed by oxidative attack on

thymine opposite adenine. Tg:G is formed by oxidative attack on 5-methylcytosine opposite

guanine (3). It is possible that the in vitro substrate selectivity of mammalian Nth1 and the

concomitant selective stimulation of activity by APE1 are indicative of selective repair of

oxidative damage in different regions of the genome.

INTRODUCTION

Like its E. coli homolog, endonuclease III (Nth), hNth1 is a bifunctional DNA N-

glycosylase/AP lyase which removes ring-saturated pyrimidines, be they hydrated, reduced or

oxidized, from the DNA backbone as the initial step of base excision repair (BER) of such

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modified residues 1. The oxidation product of thymine, 5,6-dihydroxy-5,6-dihydrothymine

(thymine glycol [Tg]) is a widely studied model substrate for hNth1, which catalyzes its release

from DNA via its DNA N-glycosylase activity. This enzymatic activity is mediated via

formation of a transient Schiff base (imino) DNA-enzyme intermediate, an example of covalent

catalysis and a characteristic of all known bifunctional DNA N-glycosylases/AP lyases (4,5).

The Schiff base moiety has been hypothesized to be required for the enzymatic catalysis of the

β-elimination reaction, which effects DNA strand cleavage 3’ to the abasic (apurinic/apyrimidinic,

AP) site formed as product of the release of the base from the 2’-deoxyribose moiety to which it

was linked. The hydrolysis of the Schiff base intermediate can occur in the absence of, or

following, enzyme-catalyzed β-elimination (the AP lyase activity), resulting in DNA strand

cleavage. The enzymatic catalysis of β-elimination by DNA N-glycosylases/AP lyases has been

shown to be initiated via abstraction of the deoxyribose pro-S-2’-hydrogen by a basic amino

acid in the enzyme active site. Several factors, including pH, can affect the efficiency of the AP

Lyase step by affecting substrate binding and proton abstraction (5).

Based on the results of studies with Nth, the 2 activities of hNth1, DNA N-glycosylase

and β-elimination catalysis, require the formation of the Schiff base intermediate and were

thought to occur concomitantly. However, data from our laboratory and from other laboratories

indicated that DNA N-glycosylase and β-elimination catalysis by mammalian members of the

endonuclease III enzyme superfamily was not concurrent under the assay conditions employed

(1, 6-8). We were the first to report that the DNA N-glycosylase activity and the AP lyase

activity of hNth1 were not concurrent i.e. that the rate of AP lyase mediated strand cleavage was

much slower than the rate of DNA N-glycosylase mediated base release. These results were

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similar to the non-concurrence of base release and strand cleavage reported for mammalian 8-

oxoguanine DNA N-glycosylase homologue, Ogg1 (8). In this report, we present data

demonstrating that the dissociation of the 2 activities of hNth1 is dependent on the nature of the

orphan base opposite the Tg residue in DNA.

In our studies of the properties of hNth1, we have focused our attention on protein

modulators of hNth1 activities. This approach resulted from our original observation that the

enzymatic properties of mammalian endonuclease III homologs were strikingly different from

bacterial endonuclease III. Our laboratory was the first to identify mammalian endonuclease III

homologs and describe their distinct kinetic properties (9, 10). These observations spurred us to

isolate novel proteins which might modulate the enzyme activities of mammalian Nth1. A yeast

2 hybrid search resulted in identification of the pluripotent transcription factor Y-box binding

protein 1 (YB-1, also known as DNA binding protein B) as a stimulator of hNth1 activity (1).

We now report further aspects of the proteomics of hNth1-initiated BER, based on the

effects of human AP endonuclease APE1/HAP-1/ref-1 (APE1) on hNth1 activity. APE1, a

human homologue of E. coli exonuclease III (Xth), catalyses the hydrolysis of the

phosphodiester bond 5’ to AP sites, generating a free 3’ hydroxyl end for the initiation of DNA

polymerase repair synthesis.

APE1 has recently been demonstrated to play a significant role in the coordination of

BER. APE1 has been shown to modulate the activities of several major DNA N-glycosylases,

although physical interaction between APE1 and most of these DNA N-glycosylases has not

been demonstrated (6, 7, 11, 12). The functional interaction between APE1 and DNA N-

glycosylases is consistent with the model of the coordinated activities of BER enzymes at sites of

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DNA damage (13-16). Two groups independently demonstrated the stimulation of hOgg1 by

APE1 (6, 7). Both groups demonstrated that APE1 replaced hOgg1 bound to the AP site

following base release and prior to hOgg1-catalysed β↑elimination. This resulted in bypass of

the AP lyase step and an increase in hOgg1 turnover. In this paper we report similar findings

with hNth1 and APE1. However, stimulation by APE1 proved to be dependent on the nature of

the orphan base opposite the Tg residue in DNA.

EXPERIMENTAL PROCEDURES

Proteins- Expression and purification of recombinant hNth1 was induced as described

previously (10). hNth1 concentration was quantified using an extinction coefficient of 1 O.D. at

A410 = 69.4 µM for the C-terminal cubane 4[Fe-S] cluster (17). YB-1 was expressed with a 6x

His tag in Trichoplusia ni cells and purified as described (1). DNA polymerase β was a gift from

S.H. Wilson (NIEHS) and APE1 was a gift from D. M. Wilson III (University of California).

FPG enzyme was purchased from New England Biolabs and UDG enzyme was purchased from

Invitrogen.

2’-Deoxyribose oligonucleotides- The 30-mer 2’-deoxyribose oligonucleotide substrate

containing a Tg residue at position 13 d(GATCCTCTAGCGTgCGACCTGCAGGCATGCA)

was prepared as described (1). The 30-mer 2’-deoxyribose oligonucleotide substrate containing

a uracil (U) residue at position 13 of the identical sequence, as well as the complementary 2’-

deoxyribose oligonucleotides were synthesized by the N.Y.U. School of Medicine Department of

Cell Biology. 2’-Deoxyribose oligonucleotides were deblocked, deprotected and purified by

20% denaturing PAGE. The 2’-deoxyribose oligonucleotides were gel purified and labeled at

the 5’-end using [γ-32P]ATP (NEN) and T4 Polynucleotide Kinase (Invitrogen) or at the 3’-

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end using [α-32P]ddATP (Amersham Biosciences) and Terminal Transferase (New England

Biolabs). All labeled nucleotides were purified by G-25 spin column filtration (Amersham

Biosciences) and annealed to the complementary strand which contained an adenine or guanine

base opposite the Tg or U residue.

Enzyme Assays- Unless otherwise indicated, assays were performed at 37°C in buffer

containing 50 mM HEPES pH 7.6, 100 mM KCl, 0.1 mg/mL BSA, 5mM MgCl2 and 1 mM

DTT. Enzyme, protein and substrate were diluted to working conditions in assay buffer and

equilibrated at 37°C. MgCl2 independent assays were performed with the above reaction buffer

recipe minus the addition of MgCl2, in the presence of 5 mM EDTA, which permits binding of

APE1 to DNA but inhibits its endonuclease activity (18). Reactions contained 40 nM [32P] 5’-

end labeled 2’-deoxyribose oligonucleotide substrate duplex, 5 nM hNth1 with or without 20

nM APE1. Two sets of 10 µL aliquots were taken at the indicated time periods and snap frozen

in ethanol and dry ice, after which one set was treated with 5 µL of 0.5 M putrescine pH 8.0 to

measure base release. The treated assay mixtures were then heated at 95°C for 5 min, followed

by the addition of 15 µL of loading dye (95% deionized formamide, 10 mM EDTA, 0.05%

Bromophenol Blue, and 0.05% Xylene Cyanol). To measure strand cleavage, samples were

treated with an equal volume of loading dye. All samples were then heated at 55°C for 5 min and

products were separated by 20% PAGE in 7M urea and 1 x TBE. Low substrate concentration

assays were performed with 20 nM hNth1 and 20 nM [32P] 5’-end labeled 2’-deoxyribose

oligonucleotide substrate duplex. At indicated times, aliquots were taken and treated as above.

Multiple turnover assays at Vmax were performed individually in 10 µL volumes containing

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reaction buffer, the indicated concentration of [32P] 5’-end labeled 2’-deoxyribose

oligonucleotide substrate duplex, and 20 nM hNth1 with or without 100 nM YB-1 and/or 100

nM APE1. The reactions were split into two 5 µL aliquots, snap-frozen in ethanol and dry ice

and treated as described above.

To discriminate between endonucleolytic and AP lyase products, reactions were

performed using [α-32P]ddAMP 3’-end labeled 2’-deoxyribose oligonucleotide substrate

duplex. Reactions contained the indicated substrate and enzyme concentrations and were

incubated at 37°C for 30 min. NaBH4 was added to 100 mM and the reactions were incubated at

37°C for 20 min for reduction of the 2’-deoxyribose 5’-phosphate moiety (19). The products

were purified by G-25 spin column filtration prior to separation and analysis.

Single turnover assays contained 10 nM [α-32P]ddAMP 3’-end labeled 2’-deoxyribose

oligonucleotide duplex and 100 nM hNth1 with or without 100 nM APE1 in a volume of 220 µL.

Enzyme reaction mixtures were incubated together for 2 min prior to initiation of the assay upon

addition of substrate. To measure base release, 5 µL aliquots were removed at indicated time

periods, snap frozen and treated with 5 µL of 0.5 M putrescine pH 8.0. The treated assay

mixtures were then heated at 95°C for 5 min, followed by the addition of 10 µL of loading dye.

To measure strand cleavage and determine incision products, 5 µL aliquots were removed at

indicated time periods, snap frozen and treated with an equal volume of 0.5 M NaBH4 at 37°C

for 20 min for the reduction of the dRP moiety. Samples were then filtered through a G-25 spin

column and treated with an equal volume of loading dye. Samples were then heated at 55°C for 5

min and products were separated by 20% PAGE in 7M urea and 1 x TBE. All products were

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analyzed quantitatively via phosphorimaging using a Molecular Imager FX System with

Quantity One Software (Bio-Rad).

Crosslinking of Enzyme to 2’-Deoxyribose Oligonucleotide Substrate (Enzyme Trapping)– For

NaBH4 reduction, assays contained 400 nM of the indicated [32P] 5’-end labeled 2’-

deoxyribose oligonucleotide duplex and 20 nM hNth1 with or without 100 nM APE1 and/or 100

nM YB-1 in a volume of 10 µL. After a 15 min incubation, reaction mixtures were treated with

5 µL of 0.5 M NaBH4 and incubated at 37°C for 5 min. After addition of an equal volume of

Laemmli SDS-loading buffer, samples were boiled in preparation for separation and analysis.

All samples were separated by 12% SDS PAGE and analyzed quantitatively via

phosphorimaging as above.

RESULTS

Differential Processing of Tg Opposite Adenine and Tg Opposite Guanine by hNth1- We

previously demonstrated, using a Tg:A-containing substrate, that the initial rate of DNA N-

glycosylase-mediated release of Tg by hNth1 was much greater than the rate of AP lyase-

mediated DNA strand cleavage (1). Since all previous assays with Tg had been done with

adenine as the opposite base, and since this may not be the only context in which Tg occurs in

vivo, we decided to look at hNth1 activity against substrates containing Tg paired opposite

guanine (2). Figure 1 is confirmation of our previous data. The assay was carried out in a shorter

reaction period than we previously used revealing that the difference between the rate of base

release and strand cleavage of a Tg:A substrate was even greater than we had reported, differing

by an order of magnitude. In sharp contrast, the AP lyase activity of hNth1 was the same as its

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DNA N-glycosylase activity against a Tg:G substrate. The DNA N-glycosylase activity of the

enzyme against Tg:A appeared to reach Vmax at substrate concentrations greater than 400 nM.

However, as we previously reported, hNth1 does not follow Michaelis-Menten pseudo-zero

order kinetics (1). Instead, the apparent kcat (which includes the rates of product release)

increased with increasing hNth1 concentration, suggesting positive cooperativity in hNth1

substrate processing. This observation and interpretation has been corroborated and expanded

upon by Liu and Roy (20). Because of this phenomenon, kcat and Km values for the duplex 2’-

deoxyribose oligonucleotide substrates vary with enzyme concentration. As we previously

suggested, the V vs. S data of Figure 1 reflect the fact that the enzyme was dissociating from the

DNA N-glycosylase generated AP:A site and initiating base release on new Tg:A substrates

without catalyzing β-elimination (1). However, when the substrate was Tg:G there was no

evidence of enzyme dissociation from AP:G (2).

APE1 Increases hNth1 Processing of Tg:A-containing 2’-Deoxyribose oligonucleotide

Substrates– We looked at the effect of APE1 on the activities of hNth1. There is evidence that

APE1 interacts with DNA N-glycosylases in BER as part of a “single-nucleotide BER relay”

involved in the coordination of processing of BER pathway intermediates (13, 21, 22).

Figure 2 shows that addition of APE1 increased hNth1 activity against a Tg:A substrate.

The stimulation of hNth1 activity was independent of the enzymatic activity of APE1. The assay

mixture lacked MgCl2 and contained sufficient EDTA to abrogate APE1-mediated

endonucleolytic cleavage but not the binding of APE1 to DNA (18). Identical experiments with

Tg:G substrates showed no increase in hNth1 substrate processing in the presence of APE1 (data

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not shown). We found that the optimum ratio for stimulation of hNth1 by APE1 varied with both

hNth1 and substrate concentration, ranging from 1:1 to 1:10. We suggest that this variability was

due, at least in part, to the positive cooperativity exhibited by hNth1 (1, 20).

APE1 Abrogates hNth1 AP Lyase Activity on Tg:A Containing Substrates But Not Tg:G

Containing Substrates- Having determined that the effect of APE1 on hNth1 activity differed

with substrate, we characterized the incision products generated by APE1 and hNth1 with the 2

substrates. The incision products generated by hNth1 in the presence of APE1 in vitro differed

with each substrate. Figure 3 is the gel analysis of the incision products of [32P] 3’-end labeled

Tg:A and Tg:G-containing 2’-deoxyribose oligonucleotide duplex substrates generated by

hNth1 in the presence or absence of APE1. In contrast to the experiments shown in Figure 2,

MgCl2 was added to the reaction to activate the endonucleolytic activity of APE1. The 2’-

deoxyribose 5’-phosphate (dRP) residue generated by APE1-catalyzed endonucleolytic

cleavage of an AP site is extremely labile and not retained during electrophoresis. Reduction by

NaBH4 prior to electrophoresis stabilizes the sugar moiety and permits size discrimination

between 5-endonucleolytic and 3-β-elimination AP lyase products (19). The AP lyase product

(p-17-p*ddA) can be seen in lanes containing hNth1 alone, with either Tg:A or Tg:G substrate

(lanes 2 and 4 respectively). Addition of APE1 to hNth1 using Tg:A produced a higher

molecular weight incision product, which is the endonucleolytic product (p-rAP-p-17-p*ddA)

generated by APE1 (lane 3). In the case of the Tg:G substrate, no change in the size of the

incision product was detected upon APE1 addition, indicating that the AP lyase activity of hNth1

was unaffected by APE1 with this substrate (lane 5). In this figure, there are 2 marker lanes for

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the β-elimination product, one with Tg:G containing substrate and one with U:A containing

substrate (lanes 6 and 8, respectively). The formamidopyrimidine-DNA N-glycosylase (FPG)

enzyme is a DNA N-glycosylase with β/δ elimination activity. Since the substrate is 3’-labeled,

the only product observed with FPG enzyme is the β-elimination product (lane 6). A second

marker lane for the β-elimination product is the AP-site containing substrate generated by

incubation of U:A-containing substrate with UDG and hNth1 to generate the AP lyase product

(p-17-p*ddA) (lane 8). The U:A-containing substrate incubated with UDG and APE1 serves as

the marker for the endonucleolytic product (p-rAP-p-17-p*ddA) (lane 7).

Incision Products Generated by hNth1 in the Presence or Absence of APE1, YB-1 and β -pol

Differ In Vitro- Since our previous work had identified YB-1 as a modifier of hNth1 activity,

we next investigated whether the addition of YB-1 together with APE1 had any effect on the

nature of the incision products of hNth1 with [32P]ddAMP 3’-end labeled Tg:A-containing 2’-

deoxyribose oligonucleotide duplex substrate (Figure 4). The addition of YB-1 in the presence

or absence of APE1 increased product formation (lanes 2 and 4). However, it did not change the

nature of the incision product, which in the presence of APE1 was the endonucleolytic product

(p-rAP-p-17-p*ddA) (lanes 3 and 4).

Since hNth1 did not demonstrate AP lyase activity against Tg:A sites in the presence of

APE1, we investigated whether the removal of the dRP residue could be effected by DNA

polymerase β (β-pol). Figure 4 demonstrates the reappearance of the β-elimination product

upon the addition of β-pol to hNth1 and APE1 with a Tg:A substrate (lane 7). This observation

is consistent with the known dRPase activity of β-pol (22). The marker lane for the β↑

elimination product (p-17-p*ddA) contains FPG enzyme (lane 9).

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Similar experiments with an N-terminal deletion mutant of hNth1, lacking amino acids

1-57, produced the same results in the presence of APE1 and YB-1 (data not shown),

suggesting that the effects of YB-1 and APE1 are not mediated by the N-terminus of hNth1.

APE1 Does Not Increase DNA N-Glycosylase Activity of hNth1 Under Conditions of Single

Turnover- To elucidate the mechanism by which APE1 affects hNth1 activity, we asked if APE1

changed the catalytic properties of hNth1 or whether it affected product inhibition. To address

this, we conducted the reaction under single turnover conditions where [E]>>[S], using

[32P]ddAMP 3’-end labeled substrates to determine the nature of the incision product. Since all

substrate molecules are bound by enzyme under single turnover conditions, the rates which

determine product formation are dependent only on the rate of base release (Schiff base

formation) and β-elimination (24). If APE1 did not affect either of these catalytic properties,

then there should be no stimulation under conditions of single turnover.

Figure 5 illustrates that no stimulatory effect of DNA N-glycosylase activity was

observed under conditions where neither enzyme turnover nor substrate binding contributed

significantly to the rate of the reaction. A complete abrogation of the AP lyase-mediated AP site

cleavage was seen with the Tg:A substrate (Figure 5A). This is consistent with the results of

Figure 3 (lane 3) and Figure 4 (lane 3).

Interestingly, in the case of the Tg:G substrate, addition of APE1 increased the rate of

hNth1 AP lyase activity (Figure 5B). We previously mentioned that addition of APE1 did not

result in increased product formation by hNth1 under turnover conditions using Tg:G. Thus, the

results of Figure 5B suggest that hNth1 remained tightly bound to the nicked AP:G DNA which

is the product of AP lyase activity.

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With both Tg:A and Tg:G substrates, the binding to the substrate and base release was

rapid, while β-elimination-catalyzed cleavage of the resulting AP site was clearly the rate

limiting reaction. Under single turnover conditions, the lag of the AP lyase step behind the DNA

N-glycosylase step was only slightly longer for Tg:A than Tg:G, indicating that the difference in

processing of the 2 substrates was due to enzyme turnover determined by the relative

dissociation rates of hNth1 from its products (Figure 5). Further evidence for this is shown in

Figure 6, in which both hNth1 and Tg:A and Tg:G substrates were present at low, equimolar

concentrations. Under these conditions, the dissociation of the DNA N-glycosylase and AP lyase

activities of hNth1 were apparent against the Tg:A substrate, but there was no dissociation

between the 2 activities against the Tg:G substrate.

APE1 Stimulates hNth1 Activity Under Conditions of Vmax- In order to determine whether

APE1 increased the maximal rate of turnover for the enzyme, we tested stimulation by APE1

under conditions of Vmax for both DNA N-glycosylase and AP lyase activities using 20 nM

hNth1 and 400 nM Tg:A substrate identical to the conditions of Figure 1. However, in the

experiments of Figure 7 MgCl2 was added to activate APE1 endonucleolytic activity. In

reactions containing hNth1 alone or hNth1 with YB-1, DNA cleavage was due solely to hNth1-

catalyzed β-elimination. For all 4 sets of experiments in this figure, DNA N-glycosylase activity

was quantified by measuring the cleavage of hNth1-generated AP sites by treatment with

organic base (putrescine) which effects cleavage via β-elimination (25). In reactions containing

APE1, all cleavage was the result of APE1 activity. Addition of APE1 resulted in a 2 fold

increase in the number of hNth1-generated AP sites as compared to hNth1 alone. Addition of

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YB-1 also caused a 2 to 3 fold increase in DNA N-glycosylase activity as we previously

described (1). Like YB-1, APE1 increased the kcat of hNth1 (as a DNA N-glycosylase) under

conditions of substrate excess. The last experiment of this group indicates that the effects of

APE1 and YB-1 were additive, resulting in a close to 4 fold increase in product. This combined

stimulation could actually be much greater, since, under our experimental conditions, most of the

substrate had been processed within 15 min.

Corroborating our results of experiments performed under low Tg:G substrate

concentrations, the addition of APE1 to hNth1 using substrate under conditions of Vmax (400

nM Tg:G) did not result in increased product formation (data not shown). Interestingly, hNth1

activity against Tg:G was stimulated by addition of YB-1, but there was no additive effect with

APE1 (data not shown).

APE1 Addition Decreases the Half Life of Covalent ES Complexes Formed by hNth1 with Its

Substrate- In order to investigate the effect of APE1 on the reaction intermediates of hNth1 with

its substrate, we used the reducing agent NaBH4, to trap the covalent ES intermediate (Figure 8).

NaBH4 is a strong reducing agent, capable of reducing the Schiff base as well as existing AP

sites (26). Therefore, the trapping of the covalent reaction intermediate with NaBH4 (Figure 8)

revealed the number of covalent complexes present in the reaction mixture at a given time point,

in this case, 15 min. For Tg:A substrate under steady state conditions, the addition of APE1

resulted in a decrease in the steady-state number of complexes initiated by hNth1 15 min after

the start of the reaction when the rate of product formation was still linear with time. There was

no significant change in the number of steady-state complexes with the Tg:G substrate upon

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APE1 addition. The addition of YB-1 resulted in a greater number of complexes with both

substrates, corroborating our previous data (1 and data not shown). The marked reduction in the

number of ES complexes (Tg:A) in the presence of both YB-1 and APE1 is a reflection of the

increased stimulation of hNth1 turnover, resulting in almost total substrate processing within 15

min. This data suggests that APE1 decreased the half-life of the enzyme-substrate covalent

intermediate, thereby stimulating dissociation of hNth1 from the AP:A site without catalyzing β↑

elimination, consistent with results of other laboratories studying the effect of APE1 on hOgg1 (6,

7).

DISCUSSION

The generation of Tg in DNA occurs through oxidative attack on the pyrimidine bases.

Tg opposite adenine can be formed by direct hydroxyl radical attack under aerobic conditions on

thymine in situ opposite its correctly paired base. As a blocker of both DNA and RNA

polymerases, Tg opposite adenine is a cytotoxic lesion (27). When Tg is bypassed by a DNA

polymerase, adenine is usually incorporated opposite Tg (27). Since the 2’-deoxynucleotide

triphosphate of Tg has been shown to be a poor substrate for polymerase incorporation,

formation of Tg:G is unlikely to be the result of insertional mispairing (28).Thus, the formation

of Tg:G is due primarily to direct oxidative attack on 5-methylcytosine resulting in deamination

and oxidation as we previously demonstrated (3). This modification is likely to occur in CpG

islands, which are rich in 5-methylcytosine and play a role in the regulation of gene expression.

In contrast to Tg:A, Tg:G is potentially mutagenic because, if unrepaired and bypassed by a

DNA polymerase, the insertion of adenine opposite the Tg would lead to a CGàTA transition

mutation as well as a disruption in methylation patterns (3).

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We here further investigated the mechanism of the difference of activities of hNth1

against Tg when it is paired with a different orphan base. While hNth1 exhibited marked

dissociation between its DNA N-glycosylase and AP lyase activities when the substrate was

Tg:A, the enzyme demonstrated concomitant DNA N-glycosylase and AP lyase activities when

the substrate was Tg:G. While the rates for base release by the enzyme were much greater for

Tg:A than for Tg:G, the rates for AP lyase activity initiated by hNth1 against Tg:A and Tg:G

were nearly identical. Our data indicate that this discrepancy was due to the presence or absence

of hNth1 turnover rather than inherent differences in reaction rates.

Furthermore, we report that APE1 stimulated hNth1 activity against Tg:A by abrogation

of the AP lyase step, presumably by effecting the dissociation of hNth1 from the AP site, thereby

increasing the turnover of the DNA N-glycosylase activity of hNth1 (Figure 9A). Thus, in the

presence of APE1, hNth1 essentially becomes a monofunctional DNA N-glycosylase against

Tg:A but not against Tg:G. Similar results have been reported hOgg1 (6, 7). While we did not

observe stimulation of hNth1 product formation by APE1 with Tg:G under turnover conditions,

we did observe APE1 stimulation of hNth1-catalyzed β-elimination with Tg:G under single

turnover conditions (Figure 5B). Taken together, this data suggests that hNth1 remains bound to

the nicked AP site product. This indicates that APE1 did not mitigate the inhibition of hNth1 by

the nicked DNA product of β-elimination of the AP:G site (Figure 9B). While we had

previously suggested Schiff base hydrolysis (k4) and product release (k5) not to be rate limiting,

our current data suggests the opposite (1). The observation that YB-1 did stimulate Tg:G

substrate processing by hNth1 suggests that YB-1 may effect product release as well as the

steady-state equilibrium between the Schiff base ES intermediate and the non-covalent ES

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intermediate.

What is a possible mechanistic reason for differential activity of hNth1 acting on Tg:A

vs. Tg:G? A detailed in vitro analysis of hNth1 substrate processing by Eide et al. revealed that

the β-elimination activity of hNth1 was strongly dependent upon the nature of the orphan base

pairing with an AP site, being a 100 fold greater with AP:G as compared to AP:A (29). Thus, the

difference in hNth1 processing of Tg:A vs. Tg:G may be a reflection of the affinity of hNth1 for

AP:G vs. AP:A. From our turnover data (Figures 1 and 6), we conclude that the AP lyase activity

of hNth1 against the AP-site containing substrate is dependent on the binding affinity of hNth1

for the AP site. Thus, the dissociation of hNth1 from the AP:A substrate following the enzymatic

release of Tg suggests a low binding affinity of hNth1 for the AP:A site. This is shown in the

kinetic mechanism outlined in Figure 9A, where k6 represents the hydrolysis of the Schiff base

without catalysis of β-elimination and k7 represents the dissociation of the enzyme from the AP

site. Our data suggests that APE1 drives the steady state equilibrium from the covalent (Schiff

base, E=S2) to the non-covalent (ES2) intermediate between hNth1 and its AP:A substrate,

resulting in the dissociation (E+S2) of hNth1 from the AP:A site. The higher affinity of hNth1

for AP:G is depicted by virtually unobservable values for k6 and k7 , in which case all Schiff

base formation results in catalysis of β-elimination and formation of AP lyase product (E=P2)

(Figure 9B). Since APE1 stimulates hNth1 AP lyase activity with Tg:G without stimulating

turnover, we suggest that hNth1 remains tightly bound to its AP lyase product (E=P2).

The different activities that we observed with hNth1 in vitro may be indicative of the

involvement of different substrate-dependent interacting and modifying proteins in vivo. Certain

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lesions, by virtue of their mutagenic or toxic characteristics, may be preferentially channeled

down a particular BER pathway i.e. long-patch or short patch BER. It has been suggested that

the intrinsic properties of the DNA N-glycosylase involved in the initial recognition and

removal of the damaged base may be responsible for BER pathway selection (30). In this case,

the intrinsic properties may include the pairing base discrimination exhibited by hNth1.

The in vivo significance of AP lyase activity is poorly understood. The removal of the

damaged base by the DNA N-glycosylase generates an AP site, which is a labile, and therefore

dangerous, intermediate for the cell. AP sites are the universal intermediates for all BER, and it is

postulated that most of these sites are processed by APE1 (31). Our studies with hNth1 are

consistent with studies performed with mammalian Ogg1, which suggested that AP site

processing is the rate-limiting step in BER initiated by bifunctional DNA N-glycosylase/AP

lyases (8). As we previously proposed, severe oxidative stress or UV irradiation would result in

the formation of a large number of damaged bases throughout cellular DNA (1). In theory, the

spontaneous removal of the damage via concomitant DNA N-glycosylase/AP lyase activity by

bifunctional DNA N-glycosylase/AP lyases would result in the simultaneous formation of a

large number of DNA strand breaks, a cytotoxic intermediate. In support of this hypothesis,

recent data on H2O2 sensitivity in E. coli identified the intermediates of Tg repair, rather than

the persistence of Tg, as significant contributors to cell death (32). The delayed AP lyase activity

and product inhibition inherent in hNth1 activity may allow for the regulation of AP site

processing. The substrate specific product inhibition exhibited by hNth1 may also represent the

channeling of AP site processing into specific BER pathways in different regions of the genome.

The rate of removal of Tg from cellular DNA has been shown to be very slow, as compared to

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the removal of uracil or the repair of AP sites in DNA and has been reported to consist mostly of

short patch BER involving β-pol (23, 33, 34). Our data suggests that it is the AP lyase activity of

β-pol that is responsible for the removal of the dRP residue left by hNth1-catalyzed base

release followed by APE1-catalyzed hydrolysis of the 5-phosphate bond. The fact that β-pol is

the primary polymerase involved in the major Tg removal pathway supports this model (34, 35).

The pivotal role played by APE1 physically connects the activities of the initial BER factors to

the DNA repair synthesis enzymes, such as β-pol, Flap endonuclease 1 (FEN-1), and DNA

ligase (13, 21, 31). Data from our laboratory and others have shown that interactions between

BER enzymes and other proteins can stimulate damage recognition or excision, suggesting the

probability of multiprotein complex formation in the initial events of BER (1, 6, 7, 36-39).

APE1 is not limited to its role in BER, but is a composite of the different activities of the

enzyme as a reduction-oxidation (redox) factor (40). Notably, APE1 has been shown to control

the activity of p53 through redox alteration and p53, in turn, has been shown to play a role in

BER (40-44). Like YB-1, APE1 has been shown to be a pluripotent factor, and may be a key

effector of the relationships between mammalian BER, oxidative signaling, transcription

regulation, and cell-cycle control (40,45).

We previously demonstrated that YB-1 affected hNth1 activity via the steady state

equilibrium between the covalent (E=S2) and non-covalent (ES2) enzyme-DNA intermediate

(1). We proposed that this equilibrium may be a checkpoint for modulation of hNth1 activity (1).

In this report, we show that APE1 modulated hNth1 activity at the same equilibrium point albeit

to a different effect. Thus, although YB-1 and APE1 seem to modulate hNth1 activity in

opposite ways, they have a synergistic effect in vitro. The overall effect on hNth1 activity by

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these 2 factors is to increase hNth1 turnover and substrate processing. Whether this has

significance in vivo remains to be determined.

In conclusion, we have performed further analyses into the mechanism of hNth1 activity

and identified substrate-specific enzymatic properties. We have also demonstrated a functional

interaction between hNth1 and the major mammalian AP endonuclease, APE1. This interaction

is dependent on the nature of the orphan base opposite Tg.

We recently reported, as has another laboratory (46), that Nth1 -/- mice are viable and

exhibit no abnormal phenotype. Extracts of tissues of Nth1-/- mice expressed an enzyme

activity that cleaved Tg:G substrate more rapidly than Tg:A substrates. This pattern seems to be

in direct contrast to the substrate selectivity of hNth1. Whether the substrate selectivity of Nth1

is a reflection of actual differences in Nth1 mediated in vivo processing of Tg:A vs. Tg:G, and

whether the selective stimulatory effects of APE1 affect processing, awaits further elucidation.

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AKNOWLEDGMENTS

D Supported by the Department of Pathology, New York University School of Medicine and by

an award from the New York University School of Medicine Research Bridging Support Fund

(G.W.T.), NIEHS grant ES 09127 (A.K.B.) and by GM 46312 (R.P.C.). We thank Dr. David M.

Wilson III for supplying APE1and Dr. Samuel H. Wilson for supplying DNA Polymerase β.

FOOTNOTES

Abbreviations:

1hNth1, human endonuclease III homologue 1; AP, apurinic/apyrimidinic; Ogg1, 8-

oxoguanine-DNA N-glycosylase; YB-1, Y-Box binding protein 1; BER, base excision repair;

SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Tg, thymine glycol;

rAP, reduced apurinic/apyrimidinic; dRP, 2’-deoxyribose phosphate; APE1,

apurinic/apyrimidinic endonuclease 1; TBE, Tris-Borate EDTA; FPG, formamidopyrimidine-

DNA N-glycosylase; UDG, Uracil DNA N-glycosylase; β-pol, DNA polymerase β.

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FIGURE LEGENDS

Figure 1. hNth1 activity against Tg:A vs. Tg:G-containing 2-deoxyribose oligonucleotide

duplex substrates. 20 nM hNth1 was incubated with specified concentrations of [32P] 5’-end

labeled 2-deoxyribose oligonucleotide duplex substrate containing either an A (circles) or G

(squares) opposite the Tg moiety. Open symbols denote DNA N-glycosylase activity determined

by putrescine treatment of reaction products. Solid symbols denote AP lyase activity. Mean

values were calculated from 2 independent experiments and did not vary by more than 10%.

Figure 2. Effect of APE1 on hNth1 activity against a Tg:A-containing 2’-deoxyribose

oligonucleotide duplex substrate. 40 nM Tg:A-containing [32P] 5’-end labeled 2’-deoxyribose

oligonucleotide duplex substrate was incubated with 5 nM hNth1 with (squares) or without

(circles) 20 nM APE1. APE1 endonucleolytic activity is abrogated in the absence of MgCl2 and

the presence of 5 mM EDTA. Open symbols denote DNA N-glycosylase activity determined by

putrescine treatment of reaction products. Solid symbols denote AP lyase activity.

Figure 3. Effect of APE1 on the incision products generated by hNth1 in vitro against Tg:A and

Tg:G–containing 2’-deoxyribose oligonucleotide duplex substrates. Reactions contained 10 nM

Tg:A or Tg:G-containing [32P]ddAMP 3’-end labeled 2’-deoxyribose oligonucleotide duplex

substrate, 20 nM hNth1 and 10 nM APE1, as indicated. Lane 6 contained 1 unit of FPG enzyme.

Another marker lane for the β-elimination product is also shown with a U-containing radio-

labeled substrate incubated with UDG and hNth1 (p-17-p*ddA, where * denotes the labeled

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phosphate). The U-containing substrate was incubated with UDG and APE1 to generate a

marker for the endonucleolytic product (p-rAP-p-17-p*ddA). Reactions were incubated at

37°C for 30 min prior to addition of NaBH4. followed by analysis as described in experimental

procedures.

Figure 4. Effects of APE1, YB-1 and β -pol on the incision products generated by hNth1 in

vitro against Tg:Acontaining 2-deoxyribose oligonucleotide substrates. Reactions contained 20

mM dNTPs, 16.6 nM Tg:A-containing [32P]ddAMP 3’-end labeled duplex 2’-deoxyribose

oligonucleotide substrate, 16.6 nM hNth1, 8.3 nM APE1, 8.3 nM β-pol and 83 nM YB-1, as

indicated. Lane 9 contained 1 unit of FPG enzyme as a marker lane for the β-elimination product

(p-17-p*ddA, where * denotes the labeled phosphate). Reactions were incubated at 37°C for 30

min prior to addition of NaBH4. followed by analysis as described in experimental procedures.

Figure 5. Representative plot of the effect of APE1 on single-turnover kinetics of hNth1 with

Tg:A (Panel A) and Tg:G–containing (Panel B) 2’-deoxyribose oligonucleotide duplex

substrates. Reactions contained 10 nM Tg:A (A) or Tg:G-containing (B) [32P]ddAMP 3’-end

labeled 2’-deoxyribose oligonucleotide substrate duplex, 100 nM hNth1 with (squares) or

without (circles) 100 nM APE1. Open symbols denote DNA N-glycosylase activity determined

by putrescine treatment of reaction products. Solid symbols denote AP lyase activity. Reactions

were incubated at 37°C for the indicated time period after which aliquots were stopped by snap

freezing. One set was treated with putrescine and the other with NaBH4.Analysis was as

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described in experimental procedures.

Figure 6. Representative plot of product formation by hNth1 against Tg:A and Tg:G-containing

2’-deoxyribose oligonucleotide duplex substrates as a function of time under multiple-turnover

conditions. Reactions contained 20 nM Tg:A (circles) or Tg:G-containing (squares) [32P] 5’-

end labeled 2’-deoxyribose oligonucleotide duplex substrate as indicated, with 20 nM hNth1.

Open symbols denote DNA N-glycosylase activity determined by putrescine treatment of

reaction products. Solid symbols denote AP lyase activity.

Figure 7. Effect of APE1 on hNth1 activity against a Tg:A-containing 2’-deoxyribose

oligonucleotide duplex under conditions of Vmax. Reactions consisted of 20 nM hNth1

incubated with 400 nM Tg:A [32P] 5’-end labeled 2’-deoxyribose oligonucleotide duplex

substrate with 100nM YB-1 and/or 100 nM APE1, as indicated, for a reaction time of 15 min.

Black bars denote DNA cleavage, either by hNth1-catalyzed AP lyase activity (in the absence of

APE1) or APE1 endonucleolytic activity (in the presence of APE1). Grey bars represent reaction

products treated with putrescine. In the absence of APE1, these denote hNth1 DNA N-

glycosylase activity. In the presence of APE1, all AP sites generated by hNth1 DNA N-

glycosylase activity were endonucleolytically cleaved by APE1. Mean values and standard

deviations were calculated from 3 independent experiments.

Figure 8. Representative plot of the effect of APE1 and YB-1 on steady state levels of hNth1

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Schiff base intermediate with Tg-containing 2’-deoxyribose oligonucleotide duplex. Trapping

of the covalent ES intermediate using NaBH4. Reactions contained 400 nM Tg:A (solid) or Tg:G

(striped)-containing [32P] 5’-end labeled 2’-deoxyribose oligonucleotide duplex substrate with

20 nM hNth1, 100 nM YB-1 and/or 100 nM APE1, as indicated. Reactions were incubated at

37°C for 15 min after which NaBH4 was added to a concentration of 100 mM and the reactions

were incubated for another 2 min at 37°C prior to the addition of Laemmli SDS sample buffer.

Figure 9. Kinetic scheme for the effect of APE1 on hNth1 Activity against Tg:A (A) and Tg:G-

containing (B) 2’-deoxyribose oligonucleotide duplex substrate.

S1 ; the substrate with modified base (i.e. Tg) intact.

S2 ; the substrate containing an AP site after base release.

P1 ; the 3’ end of the β-eliminated 2’-deoxyribose oligonucleotide.

P2 ; the 5’ end of the β-eliminated 2’-deoxyribose oligonucleotide.

= ; the Schiff base between E and S.

k2 ; the rate of base release

k3 ; the rate of β-elimination

k4 ; the rate of Schiff base hydrolysis

k-6 and k6 ; the steady state of Schiff base resolution

k-7 and k7 ; the binding and release of the non-covalently bound AP site moiety.

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(A) The inhibition of k3 by APE1 drives the reaction towards k6/k7, the dissociation of hNth1

from the AP:A substrate, which results in increased turnover, a faster reaction rate and a shorter

half-life for the covalent ES intermediate. This leaves an AP site ready for processing by APE1

and repair synthesis. (B) The effect of APE1 on k3 results in an increased rate of AP lyase

activity. However, there is no evidence that the hNth1 is driven to dissociate from the β-

elimination product and turnover is not significantly affected.

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Boorstein, Richard P. Cunningham and George W. TeeborDina R. Marenstein, Michael K. Chan, Alvin Altamirano, Ashis K. Basu, Robert J.

endonuclease 1 (APE1) on hNth1 activitySubstrate specificity of human endonuclease III (hNth1): Effect of human AP

published online January 8, 2003J. Biol. Chem.

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