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Proc Natl Acad Sci U S A. 1996 November 26; 93(24): 13737–13741. | PMCID: PMC19409 |
Copyright © 1996, The National Academy of Sciences of the USA Biochemistry An unusual mechanism for the major human apurinic/apyrimidinic
(AP) endonuclease involving 5′ cleavage of DNA containing a
benzene-derived exocyclic adduct in the absence of an  AP site B. Hang, A. Chenna, H. Fraenkel-Conrat, and B. Singer* Life Sciences Division, Donner Laboratory, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720 Accepted September 4, 1996. The major human apurinic/apyrimidinic (AP) endonuclease (class
II) is known to cleave DNA 5′ adjacent to an AP site, which is probably
the most common DNA damage produced hydrolytically or by
glycosylase-mediated removal of modified bases.
p-Benzoquinone (pBQ), one of the major benzene
metabolites, reacts with DNA to form bulky exocyclic adducts. Herein we
report that the human AP endonuclease directly catalyzes incision in a
defined oligonucleotide containing
3,N4-benzetheno-2′-deoxycytidine (pBQ-dC)
without prior generation of an AP site. The enzyme incises the
oligonucleotide 5′ to the adduct and generates 3′-hydroxyl and
5′-phosphoryl termini but leaves the pBQ-dC on the 5′ terminus of the
cleavage fragment. The AP function of the enzyme is not involved in
this action, as no preexisting AP site is present nor is a DNA
glycosylase activity involved. Nicking of the pBQ-dC adduct also leads
to the same “dangling base” cleavage when two Escherichia
coli enzymes, exonuclease III and endonuclease IV, are used.
Our finding of this unusual mode of action used by both human and
bacterial AP endonucleases raises important questions regarding the
requirements for substrate recognition and catalytic active site(s) for
this essential cellular repair enzyme. We believe this to be the first
instance of the presence of a bulky carcinogen adduct leading to this
unusual mode of action. Keywords: DNA repair, human
carcinogen, 3,N4-benzetheno-2′-deoxycytidine, dangling
base The integrity of the human genome is constantly under attack by
countless exogenous and endogenous DNA-damaging agents.
This is clearly a major threat to the cell. To cope with this
challenge, some cellular enzymes are able to recognize a wide range of
substrates and/or have bi- or multiple functions toward different
type of substrates. Benzene is an ubiquitous human carcinogen and recognized as a major
cause of acute myeloid leukemia and solid tumors as well, apparently
resulting from widespread exposure to cigarette smoke and automobile
exhaust ( 1). Benzene needs to be metabolized to exert its effects and
one pathway ( 2) leads to p-benzoquinone (pBQ), the most
potent mutagen of 12 structurally related simple benzoquinones ( 3).
p-BQ is also recognized as an animal carcinogen ( 2). Recently, we reported ( 4) that HeLa cells contained a repair activity
for 3, N4-benzetheno-2′-deoxycytidine (pBQ-dC)
and 1, N6-benzetheno-2′-deoxyadenosine (pBQ-dA)
and we have now purified to apparent homogeneity a 38.1-kDa enzyme from
human cells that specifically incises a defined oligonucleotide
containing a pBQ-dC adduct derived from the reaction of pBQ with dC
under mild conditions. We now find that the repair activity also
cleaves apurinic/apyrimidinic (AP)-containing oligonucleotides, which
raised the question regarding its relationship to the previously
described major human AP endonuclease. Such types of multifunctional
enzymes are present in both prokaryotic and eukaryotic cells ( 5, 6). In
Escherichia coli, exonuclease III represents 80–90% of the
total cellular AP activity, while endonuclease IV represents 5–10%.
Both genes have been cloned and sequenced. Recently, the crystal
structure of exonuclease III has been reported ( 7). The human AP endonuclease [variously called Ape ( 8), HAP1 ( 9), or APEX
( 10)] is quite similar to E. coli exonuclease III in terms
of protein sequence and function. The main function of the human enzyme
is 5′ AP endonuclease activity, although a DNA 3′-diesterase and RNase
H activities are also reported ( 5, 6). The human protein has also
evolved a novel function (Ref-1) capable of regulating the activity of
the AP-1 transcription factor (Jun–Fos heterodimer), which in turn
controls expression of a number of genes ( 11, 12). The relationship
between the repair activity and this redox activity is not clear. Herein we report, on the basis of sequence homology and substrate
specificities, that pBQ-dC nicking activity is, to our knowledge, a
previously undescribed function of the human AP endonuclease in both
HeLa and human leukemia HL-60 cells. The enzyme directly incises the
oligonucleotide 5′ to the pBQ-dC adduct without prior generation of an
AP site, resulting in the pBQ-dC left as a “dangling base” on the
5′ terminus. This unusual mechanism is also utilized by E.
coli AP endonucleases, exonuclease III and endonuclease IV, in the
incision of an intact oligonucleotide containing a single pBQ-dC
adduct. Summary of Purification of pBQ-dC Endonuclease from HeLa and HL-60
Cells. The detailed procedure of protein purification will be
described in a separate publication. Briefly, the cell-free extracts
after ammonium sulfate precipitation were desalted and then passed
through a Whatman phosphocellulose P11 column. Purification by multiple
column chromatography and FPLC fractionation was monitored by cleavage
of a 5′ 32P-end-labeled pBQ-dC-containing 25-mer, annealed
to a complementary strand with deoxyguanosine opposite pBQ-dC. Oligonucleotide Substrates and Markers. The synthesis of
3, N4-pBQ-deoxycytidine, its phosphoramidite and
site-directed 25-mer-defined oligonucleotide was as described by Chenna
and Singer ( 13). The sequence used in this study was
5′-CCGCTAG-pBQC-GGGTACCGAGCTCGAAT-3′. A uracil-containing oligomer used
as a control was synthesized by placing the dU at the same position
(position 8 from the 5′ end) in the same 25-mer sequence. Each of the
oligomers was annealed to the same complementary 25-mer
oligonucleotide. The 5-, 7-, 17-, and 18-mer size markers with identical sequences to
the putative cleavage products from both the dU and pBQ-dC oligomers
were synthesized and purified by OPC cartridges (Applied Biosystems).
The corresponding 5′-phosphorylated 17- and 18-mer markers were
obtained using T4 polynucleotide kinase (United States Biochemical) and
unlabeled ATP. These markers were finally purified through denaturing
PAGE ( 14). The unphosphorylated 18-mer marker containing pBQ-dC on the 5′ terminus
was synthesized using the phosphoramidite technique as described ( 13).
The presence of pBQ-dC in the oligomer was confirmed by HPLC and UV
spectra of the peak corresponding to pBQ-dC ( 13). The 5′- or 3′-End Labeling. The 25-mer oligonucleotides with
modified bases were 5′-end-labeled with [γ- 32P]ATP
(specific activity 6000 Ci/mmol; 1 Ci = 37 GBq; Amersham) and
subsequently annealed to the complementary oligonucleotide as described
by Rydberg et al. ( 15). The 3′-end labeling of
oligonucleotides or 17-mer and 18-mer markers was carried out using the
DNA 3′-end-labeling kit (Boehringer Mannheim) and
2′,3′-dideoxyadenosine 5′-[α- 32P]triphosphate (ddATP)
(specific activity 3000 Ci/mmol, Amersham) by following
manufacturer’s instructions. The 3′-end-labeled oligonucleotides were
then purified using denaturing PAGE in 18% gels. The full-length bands
were cut out and eluted overnight at room temperature with 0.3 M sodium
acetate (pH 7.25). The eluent was passed through a Sep-Pak column
(Waters) and the collected purified oligonucleotides were then annealed
to the complementary strand as described above. Protein Microsequencing. The pBQ-dC enzyme that was earlier
well separated from other proteins on a 12% precast mini-SDS
tris-glycine PAGE (Bio-Rad), was stained with a dye solution containing
Coomassie blue (0.1%). Sequencing of two internal tryptic
peptides (13 and 20 amino acids, respectively) of the 38.1-kDa target
band (Fig. ) was carried out by the Protein
Structure Laboratory, University of California, Davis, using automated
Edman degradation and subsequent identification of the
phenylthiohydantoin amino acid derivatives by HPLC.
DNA Nicking Assay. The nicking assay in this work was
performed essentially as described by Rydberg et al. ( 15,
16). The nicking reaction was carried out in a total volume of 10 μl
in 25 mM Hepes·KOH, pH 7.8/0.5 mM EDTA/0.5 mM
dithiothreitol (DTT)/0.5 μg of poly(dI-dC)/0.5 mM
spermidine/1 mM MgCl 2/250 μg of BSA/10% glycerol
for 1 hr at 37°C. Usually up to 2.5 μl (33 ng) of homogeneous
pBQ-dC enzyme was used. As the result of a generous gift of Ape,
recombinant Ape proteins ( 17), E. coli endonuclease IV from
Bruce Demple (Harvard University), a comparison could be made of
substrate specificity. In addition, exonuclease III (GIBCO/BRL) was
also used in this assay. This is illustrated in Fig.
.
End-Group Determination After Enzymatic Cleavage. To determine
the nature of the 5′-end group of the nick, 5′-end-labeled
pBQ-dC-oligomer was reacted with pBQ-dC enzyme as described above and
subsequently treated with 10 units of terminal
deoxynucleotidyltransferase (United States Biochemical) and 2 μM
unlabeled ddATP in a buffer containing 0.5 mM CoCl2 for 30
min at 37°C. Reactions were stopped and electrophoresed as described
above with appropriate markers. Similarly, to examine the 3′-end group of the nick, the pBQ-dC
enzyme-treated 3′-end-labeled pBQ-dC oligomer was incubated with 0.25
unit of calf intestinal phosphatase (Pharmacia) for 20 min at 37°C
and then run on 18% denaturing PAGE with various 3′-end-labeled size
markers. Substrate Specificity of Human Cell Preparations. The original
premise was that we were dealing with a specific DNA glycosylase since
the nicking pattern (5′ to the adduct) was the same when pBQ-dC was the
adduct as when a series of human glycosylase substrates were tested
using crude HeLa extracts. As purification proceeded, separation was
achieved between glycosylases cleaving dU, dI, ![[var epsilon]](/corehtml/pmc/pmcents/epsiv.gif) A, C, thymine
glycol, G/T mismatch, and an activity cleaving pBQ-dC. Thus, it
appeared that pBQ-dC incision was due to a distinct enzymatic activity.
Fig. shows partial or complete separation of
several of these activities on a Mono S column. At this stage of
purification, it was apparent from complete separation of activity that
A and C were repaired by separate glycosylases ( 18).
pBQ-dA nicking activity was also identified in cell-free HeLa extracts
( 4) but became marginal during attempted purification. This activity
was found to be heat-unstable compared with pBQ-dC activity and is
presently considered to be a different enzyme and is under
investigation. Identification of the Purified pBQ-dC Nicking Enzyme With the Human
AP Endonuclease. After purification of pBQ-dC nicking activity to
apparent homogeneity (Fig. ), microsequencing was feasible and two
internal tryptic peptides of 13 and 20 amino acids were found to be
identical with peptides of the major human AP endonuclease (Fig.
and refs. 8, 9, 10). This was evidence that the
pBQ-dC enzyme was the same as the human AP endonuclease since no other
protein in the protein database except eukaryotic AP endonucleases
showed absolute homology to the two peptides sequenced.
With both the purified human AP endonuclease and the recombinant human
AP proteins, we were also able to compare the nicking activity toward
pBQ-dC of these characterized human AP endonuclease and the recombinant
AP proteins, as well as two E. coli AP endonucleases,
exonuclease III and endonuclease IV. The data obtained indicated that
all the enzymes used were able to efficiently cleave the pBQ-dC
oligomer 5′ to the adduct (Fig. ). Mechanism of Cleavage of pBQ-dC by AP Endonucleases. The
accepted mechanism of AP endonucleases is to recognize an AP site,
regardless of its origin, and then cleave the phosphodiester bond 5′ to
the AP site. To examine the possible involvement of a DNA glycosylase
activity that could be either associated or coeluted with the purified
enzyme and released the pBQ residue, resulting in an AP site, 3′-end
labeling was carried out. In Fig. ,
3′-end-labeled oligomers were used to examine the nature of the 3′
cleavage product from the nick. The 3′ pBQ-dC cleavage product (Fig.
Left, lane 8) migrated slower in the gel than both
unphosphorylated and 5′-phosphorylated 18-mer markers (lanes 5 and 6).
In contrast, when a 25-mer with dU placed at the same position as
pBQ-dC was used as substrate for uracil DNA glycosylase (UDG,
GIBCO/BRL), the electrophoretic mobility of the cleavage fragment
(lane 2) is identical to that of the phosphorylated 17-mer (lane 4).
The latter is consistent with the established mechanism of action of
simple glycosylases. The addition of a 5′ AP endonuclease to the UDG
reaction did not change the UDG-mediated cleavage pattern on the gel
(data not shown).
Thus, the unexpected mobility pattern of the pBQ-dC enzyme-mediated
cleavage product seen in Fig. strongly
suggested that the pBQ-dC still remained on the 5′ end of the cleavage
product and was due to a unique mechanism that left a 5′-terminal
“dangling base.” This was confirmed by synthesizing a
pBQ-dC-containing 18-mer as a marker as shown in Fig.
Right. The 32P-3′-end-labeled cleavage fragment
from the 25-mer (lane 3) comigrated only with the pBQ-dC-18-mer marker
(lane 4) and not with the unmodified 18-mer either with or without
5′-terminal phosphate (lanes 5 and 6).
The 3′-end labeling also showed that the nicking of the pBQ-dC adduct
by E. coli endonuclease IV and exonuclease III led to the
same “dangling base” cleavage (data not shown). Determination of End Groups. The determination of end groups
at the nick was carried out using 5′- or 3′-end-labeled oligomers and
modifying enzymes. The treatment of the 5′-end-labeled cleavage product
with terminal deoxynucleotidyltransferase and ddATP resulted in a DNA
band that migrated to a position corresponding to an 8-mer, one
nucleotide longer than the cleavage fragment, indicating the presence
of a hydroxyl group at the 3′ terminus of the cleavage product (Fig.
). The presence of a 5′-terminal phosphoryl group in the 3′-end-labeled
cleavage product was confirmed by treatment of the cleavage product
with calf intestinal phosphatase, which removes a 5′-terminal
phosphoryl group, producing a hydroxyl-containing terminus. The
addition of calf intestinal phosphatase resulted in a shift of the
cleavage band to a slight slower position, indicating the presence of
the phosphoryl group, as expected (Fig. ). The major AP endonuclease is a multifunctional enzyme with its
primary biological role as a class II AP endonuclease ( 5, 6). Herein we
describe a previously undescribed activity of the enzyme acting on a
bulky exocyclic DNA adduct produced by the reaction of pBQ with dC.
This activity appears to be specific for pBQ-dC, as a series of DNA
lesions were tested and found not to be substrates for the enzyme. In this work we demonstrated that the purified pBQ-dC endonuclease and
the major human AP endonuclease appears to be identical. ( i)
Microsequencing data show complete homology between the two internal
sequences tested in this work and the corresponding peptide sequences
of the human AP endonuclease. ( ii) Homogeneous pBQ-dC
endonuclease possesses an 5′ AP activity. ( iii) The purified
Ape is also able to incise pBQ-dC adduct. ( iv) Recombinant
human Ape proteins incise the pBQ-dC adduct. Note that these proteins
were purified through two different approaches ( 17). ( v)
Both enzyme preparations have same molecular weight (within the range
of standard error). The major finding is that the pBQ-dC nicking activity/Ape is unusual
since no AP site is measurable prior to the treatment of oligomer with
the enzyme. This is supported by the study on the stability of pBQ-dC
as a nucleotide and after incorporation into oligomers ( 13). No
chemical cleavage of the pBQ-dC containing oligomer was observed after
denaturing PAGE involving heat and alkali, which normally breaks the AP
site by β-elimination. The likelihood that a DNA glycosylase activity
is involved that releases the pBQ residue, resulting in an AP site, can
be ruled out by the findings from 3′-end labeling as described in the
Results. All the data presented herein indicate that the
enzyme acts as a DNA endonuclease and incises the oligonucleotide 5′ to
the adduct, generating 3′-hydroxyl and 5′-phosphoryl termini. However,
pBQ-dC is still attached to the 5′ terminus of the cleavage fragment,
for which we use the term, “dangling base.” A schematic
representation of the mechanism of action is summarized in Fig. . Finally, this unusual mode of initiation of repair of a bulky
carcinogen-derived adduct by both human and E. coli AP
endonucleases raises new questions regarding factors in
enzyme–substrate recognition. The structural requirements for this
mechanism of enzyme action are presently under investigation using a
variety of related exocyclic carcinogen adducts. The HL-60 cells were a gift from the Cell Culture Center,
Endotronics, Inc. Minneapolis, MN, a National Institutes of
Health-sponsored facility. This work was supported by Grants CA 47723
and ES 07363 from the National Institutes of Health and was
administered by Lawrence Berkeley National Laboratory under Department
of Energy Contract DE-AC03-76SF00098. 1. International Agency for Research on Cancer. IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans. 1987; 1987. (International Agency for Research on CancerLyonFrance)Suppl. 7120122. 2. Huff J E, Haseman J K, DeMarini D M, Eustis S, Maronpot R R, Peters A C, Persing R L, Chrisp C E, Jacobs A C. Environ Health Perspect. 1989;82:125–163. [PubMed] 3. Hricko A. Environ Health Perspect. 1994;102:276–81. [PubMed] 4. Chenna A, Hang B, Rydberg B, Kim E, Pongracz K, Bodell W J, Singer B. Proc Natl Acad Sci USA. 1995;92:5890–5894. [PubMed] 5. Demple B, Harrison L. Annu Rev Biochem. 1994;63:915–948. [PubMed] 6. Barzilay G, Hickson I D. BioEssays. 1995;17:713–719. [PubMed] 7. Mol C D, Kuo C-F, Thayer M M, Cunningham R P, Tainer J A. Nature (London). 1995;374:381–386. [PubMed] 8. Demple B, Herman T, Chen D S. Proc Natl Acad Sci USA. 1991;88:11450–11454. [PubMed] 9. Robson C N, Hickson I D. Nucleic Acids Res. 1991;19:5519–5523. [PubMed] 10. Seki S, Hatsushika M, Watanabe S, Akiyama K, Nagao K, Tsutsui K. Biochim Biophys Acta. 1992;1131:287–299. [PubMed] 11. Xanthoudakis S, Miao G, Wang F, Pan Y C, Curran T. EMBO J. 1992;11:3323–3335. [PubMed] 12. Walker L J, Robson C N, Black E, Gillespie D, Hickson I D. Mol Cell Biol. 1993;13:5370–5376. [PubMed] 13. Chenna A, Singer B. Chem Res Toxicol. 1995;8:865–874. [PubMed] 14. Maniatis T, Fritsch E F, Sambrook J. Molecular Cloning: A Laboratory Mannual. 2nd Ed. Plainview, NY: Cold Spring Harbor Lab. Press; 1989. 15. Rydberg B, Dosanjh M K, Singer B. Proc Natl Acad Sci USA. 1991;88:6839–6842. [PubMed] 16. Rydberg B, Qiu Z-H, Dosanjh M K, Singer B. Cancer Res. 1992;52:1377–1379. [PubMed] 17. Wilson D M, III, Takeshita M, Grollman A P, Demple B. J Biol Chem. 1995;270:16002–16007. [PubMed] 18. Hang B, Chenna A, Rao S, Singer B. Carcinogenesis. 1996;17:155–157. [PubMed] 19. Robson C N, Milne A M, Pappin D J C, Hickson I D. Nucleic Acids Res. 1991;19:1087–1092. [PubMed] 20. Seki S, Akiyama K, Watanabe S, Hatsushika M, Ikeda S, Tsutsui K. J Biol Chem. 1991;266:20797–20802. [PubMed]
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