Báo cáo khoa học: Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA pptx

MINIREVIEW
Roles of base excision repair subpathways in correcting
oxidized abasic sites in DNA
Jung-Suk Sung
1
and Bruce Demple
2
1 Department of Life Science, Dongguk University, Seoul, South Korea
2 Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, USA
Genetic stability is threatened by the continuous
assault on cellular DNA by various reactive species of
both endogenous and exogenous origins. The most
common types of DNA damage are associated with
DNA base alteration. A well-characterized DNA base
modification is uracil, which can arise in genomic
DNA by misincorporation of dUMP during DNA syn-
thesis, or by the spontaneous deamination of cytosine
in G : C base pairs to form a premutagenic lesion
[1,2]. Reactive oxygen species, the products of normal
cellular respiration, also generate a variety of oxidized
DNA base damages, including an 8-oxoguanine that is
frequently used as a biomarker for oxidative DNA
damage [3,4]. Enzymatic methylation of DNA bases,
predominantly cytosines, plays an important role in
gene regulation, but nonenzymatic alkylation from
endogenous sources forms cytotoxic and mutagenic
products, such as 3-alkyladenine and O
6
-alkylguanine
[5,6]. Metabolic by-products (such as epoxyaldehydes),
produced during cellular lipid peroxidation, are

Keywords
2-deoxyribonolactone; DNA polymerase
beta; DNA–protein crosslinks; FEN1 protein;
long-patch BER; oxidized abasic sites;
short-patch BER
Correspondence
B. Demple, Department of Genetics and
Complex Diseases, Harvard School of Public
Health, Boston, MA 02115, USA
Fax: +1 617 432 0377
Tel: +1 617 432 3462
E-mail:
(Received 12 December 2005, accepted
6 February 2006)
doi:10.1111/j.1742-4658.2006.05192.x
Base excision DNA repair (BER) is fundamentally important in handling
diverse lesions produced as a result of the intrinsic instability of DNA or
by various endogenous and exogenous reactive species. Defects in the BER
process have been associated with cancer susceptibility and neurodegenera-
tive disorders. BER funnels diverse base lesions into a common intermedi-
ate, apurinic ⁄ apyrimidinic (AP) sites. The repair of AP sites is initiated by
the major human AP endonuclease, Ape1, or by AP lyase activities associ-
ated with some DNA glycosylases. Subsequent steps follow either of two
distinct BER subpathways distinguished by repair DNA synthesis of either
a single nucleotide (short-patch BER) or multiple nucleotides (long-patch
BER). As the major repair mode for regular AP sites, the short-patch BER
pathway removes the incised AP lesion, a 5¢-deoxyribose-5-phosphate moi-
ety, and replaces a single nucleotide using DNA polymerase (Polb). How-
ever, short-patch BER may have difficulty handling some types of lesions,
as shown for the C1¢-oxidized abasic residue, 2-deoxyribonolactone (dL).

Recent work indicates that dL is processed efficiently by Ape1, but that
short-patch BER is derailed by the formation of stable covalent crosslinks
between Ape1-incised dL and Polb. The long-patch BER subpathway
effectively removes dL and thereby prevents the formation of DNA–protein
crosslinks. In coping with dL, the cellular choice of BER subpathway may
either completely repair the lesion, or complicate the repair process by
forming a protein–DNA crosslink.
Abbreviations
AP, apurinic ⁄ apyrimidinic; BER, base excision DNA repair; DPC, DNA–protein crosslink; dL, 2-deoxyribonolactone; 5¢-dLp, 5¢-terminal dL-5-
phosphate residues; 5¢-dRp, 5¢-deoxyribose-5-phosphate; MEF, mouse embryonic fibroblasts; 5-MF, 5-methylene-2-furanone; PARP-1,
poly(ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; Polb, DNA polymerase b.
1620 FEBS Journal 273 (2006) 1620–1629 ª 2006 The Authors Journal compilation ª 2006 FEBS
reactive to DNA and give rise to covalently modified
etheno-adducts involving all four DNA bases [7].
Although the reported endogenous levels of each type
of base lesion vary among tissues and with the method
of detection, their mutagenic and cytotoxic potential
suggests that they must be considered as factors in the
induction of cancer and other diseases. Beyond this
endogenous burden of DNA damage, exposure of cells
to exogenous reactive chemical agents, derived from
environmental sources or delivered deliberately as che-
motherapeutic drugs, may directly produce further
DNA damage or modulate cellular conditions to
increase the level of damage indirectly (e.g. by disrupt-
ing mitochondrial function).
Perhaps the most important cellular defense mechan-
ism that evolved to avert the deleterious effects of the
most frequent damaged or inappropriate bases in
DNA is base excision DNA repair (BER) [8–10]. The

initial step of BER involves enzymatic activities that
process the N-glycosylic bonds linking the target bases
and their deoxyribose sugars. The first such enzyme
discovered was bacterial uracil-DNA glycosylase [11].
Subsequently, uracil-DNA glycosylases were found to
be widely distributed, and DNA glycosylases acting on
other diverse lesions (alkylated, oxidized, or photo-
damaged bases, as well as certain undamaged but
mispaired bases) have been found and characterized
for their biochemical properties and biological roles in
BER : mammalian cells contain at least 10 distinct gly-
cosylase activities [12,13]. The initial product of a
DNA glycosylase is an abasic [apurinic ⁄ apyrimidinic
(AP)] site in DNA, which is the central intermediate
during BER. AP sites can also arise spontaneously at
a substantial rate and are expected to be one of the
most frequent lesions in DNA (Fig. 1A). It has been
estimated that AP site formation through the sponta-
neous hydrolytic loss of purines generates some 10 000
AP sites per day in a mammalian cell [14,15]. Com-
bined with the AP sites produced by DNA glycosylas-
es, the daily burden of AP sites is probably much
higher. One estimate yielded steady-state levels of
50 000–200 000 AP sites per cell in various rat tissues
and human liver [16], although that seems likely to be
an overestimate [12]. AP sites are dangerous lesions
that block normal DNA replication, with cytotoxic
and mutagenic consequences [17].
Oxidative damage to DNA, mediated by free radi-
cals and reactive oxygen species, produces structurally

distinct abasic sites, known as oxidized abasic sites.
Oxidized abasic sites include lesions at DNA strand
breaks, such as 3¢-phosphoglycolate esters and abasic
residues in an uninterrupted phosphodiester backbone.
These types of DNA lesions are formed by the action
of various physical and chemical agents, including
UV and c-irradiation, heterocyclic N-oxides of the
tirapazamine family, organometallic oxidants and
the anticancer antibiotics (such as neocarzinostatin) of
the ene-diyne family [18–21]. The formation of oxid-
ized AP sites is initiated by the reaction of free radicals
with the deoxyribose sugar components of DNA and
subsequent chemical rearrangements that are modula-
ted by the presence of molecular oxygen [22,23]. The
earliest identified X-ray damage in DNA was a C1¢-
oxidized abasic lesion, 2-deoxyribonolactone (dL) [24],
which is generated by initial hydrogen abstraction
from the deoxyribose C1¢ carbon, followed by O
2
addi-
tion and base loss (Fig. 1B). Successive b- and d-elimi-
nations of dL residues yields a strand break with
3¢- and 5¢-phosphate ends and liberates 5-methylene-2-
furanone (5-MF) (Fig. 1B). 5-MF has been employed
as a characteristic product of dL in its detection in
DNA [25,26]. As determined by comparing the release
of 5-MF with concomitant DNA breakage, dL lesions
may account for up to 72% of the total sugar damage
in the irradiated DNA in vitro [25]. Comparison of the
rate of spontaneous strand scission at dL sites to the

regular (aldehyde) AP sites shows that cleavage at dL
sites is 12- to 55-fold faster than at AP sites [27]. How-
ever, the immediate breakage of DNA at the dL lesion
would not be expected under physiological conditions.
OPO
3
O
N
O
OH
AP site
Spontaneous
Base Loss
Removal of Bases
by DNA
Glycosylases
OPO
3
OPO
3
OPO
3
OPO
3
A
C1´-Oxidation
O
O
2-Deoxyribonolactone
O

OPO
3
OPO
3
OPO
3
OPO
3
OPO
3
+
O
O
5-methylene-2-furanone
N
2-
2-
+
B
Fig. 1. Abasic DNA damage. Formation of a regular abasic apurinic ⁄
apyrimidinic (AP) site (A) and an oxidized abasic site, 2-deoxyribono-
lactone (dL) (B).
J S. Sung and B. Demple BER of oxidized abasic sites
FEBS Journal 273 (2006) 1620–1629 ª 2006 The Authors Journal compilation ª 2006 FEBS 1621
The half-life of dL for spontaneous cleavage under
simulated physiological conditions was estimated to be
32–54 h in duplex DNA [28]. Recent understandings
of the chemical properties of dL indicates that these
lesions are probably subjected to cellular DNA repair
or translesion DNA synthesis, rather than directly con-

tributing to the formation of DNA strand scission.
Short- and long-patch BER in
mammalian cells
A simplified version of BER for AP sites can be des-
cribed as follows: (a) enzymatic incision of the AP site;
(b) excision of the cleaved AP site at the single-strand
break; (c) repair DNA synthesis; (d) ligation of the
nick in DNA. In mammalian cells, the major AP endo-
nuclease, Ape1 (also called Apex, HAP1, or Ref-1),
hydrolyzes the 5¢ phosphodiester bond of the AP site
to generate a DNA repair intermediate that contains a
single strand break with 3¢-hydroxyl and 5¢-deoxy-
ribose-5-phosphate (5¢-dRp) termini [29,30]. Further
repair is achieved through at least two distinct BER
subpathways that involve different subsets of enzymes,
and which result in the replacement of one nucleotide
(short-patch BER), or two or more nucleotides (long-
patch BER) (Fig. 2).
In mammalian short-patch BER, the major 5¢-dRp
excision is attributable to DNA polymerase b (Polb).
The dRp excision involves a lyase activity in the Polb
8 kDa N-terminal domain acting through a covalent,
Schiff base intermediate [31,32] (Fig. 3A). Single-
nucleotide gap-filling DNA synthesis is associated with
the DNA polymerase activity of Polb, which therefore
plays dual roles in short-patch BER. In an earlier
study, the simplest form of short-patch BER of uracil
was reconstituted in vitro by using purified human pro-
teins, including Ung, Ape1, Polb and DNA ligase III
[33]. Similar in vitro reconstitution experiments for the

repair of other base lesions or the AP site also sugges-
ted essential roles of Polb in the short-patch BER
pathway [34–36]. Involvement of Polb in the short-
patch BER of various types of DNA lesions has been
demonstrated by using cell extracts from wild-type and
Polb null mouse embryonic fibroblasts (MEF) cells
[37–40]. Some short-patch BER is still observed with
Polb-deficient cell extracts, however, which suggests
5'-P
3'-blocking
group
3'-OH
5'-dRP
3'-OH
5'-P
3'-OH
5'-P
3'-OH
5'-P
FEN1-PCNA
Polβ
and/or
Polδ/ε-PCNA
Base damage
AP site
Monofunctional
DNA glycosylase
Bifunctional
DNA glycosylase
Ape1

Ape1 / PNK
Polβ
Polβ
LIG1
LIGIII-XRCC1
(1 nt patch)
(≥2 nt patch)
Fig. 2. Short- and long-patch base excision DNA repair (BER) path-
ways. The steps involved in both pathways are discussed in the
text.
O
OPO
3
OPO
3
2-
OH
H
2
N
OH
OPO
3
H
HN
+
β-elimination
A
K72
5'-dRP lyase

K72
Polβ
OPO
3
2-
OPO
3
2-
Polβ
O
OPO
3
O
OH
OPO
3
O
HN
B
H
2
N
K72
K72
OPO
3
2-
OPO
3
2-

O
OPO
3
O
H
2
N
OH
OPO
3
O
HN
AP Lyase
C
OPO
3
2-
OPO
3
2-
Polβ
Polβ
5'-dRP lyase
Fig. 3. Excision of an abasic apurinic ⁄ apyrimidinic (AP) site and
formation of a 2-deoxyribonolactone (dL)-mediated DNA–protein
crosslink. (A) Repair of a 5¢ incised AP site, a 5¢-deoxyribose-5-phos-
phate residue (5¢-dRp) by the dRp lyase activity of DNA polymerase
b (Polb). (B) Covalent trapping of Polb by a 5¢ incised dL residue
through the dRp lyase active site of the enzyme. (C) Covalent trap-
ping of a glycosylase-AP lyase by an uncleaved dL residue.

BER of oxidized abasic sites J S. Sung and B. Demple
1622 FEBS Journal 273 (2006) 1620–1629 ª 2006 The Authors Journal compilation ª 2006 FEBS
that there is functional redundancy at the level of
DNA polymerases to provide cells with backup sys-
tems [41–43]. Despite this possibility, Polb is encoded
by an essential gene, the deletion of which causes
embryonic lethality in mice [44]. Polb-deficient MEFs
exhibit hypersensitivity to DNA alkylating agents that
require BER [44]. Somewhat surprisingly, near-normal
resistance could be restored in MEFs by providing
only the N-terminal dRp lyase domain of Polb [45],
which suggests greater functional redundancy for BER
repair polymerase activities than for dRp excision.
The long-patch BER pathway involves strand dis-
placement repair synthesis of at least two nucleotides,
with excision of the 5¢ -dRp residue as part of a flap
oligonucleotide cleaved by the FEN1 nuclease [34,46].
The identity of the polymerases involved in the long-
patch BER pathway is not yet fully understood. It has
been suggested that Polb may be responsible for the
initiation of strand displacement synthesis [40,47]. In
addition, the involvement of other DNA polymerases,
such as Pold and Pole, in long-patch BER has been
suggested [43,48,49]. A reconstituted enzyme system
was developed for long-patch BER of a reduced AP
site utilizing purified Ape1, Polb, Pold, proliferating
cell nuclear antigen (PCNA), FEN1 and DNA ligase I,
where Pold substituted for Polb when PCNA was pre-
sent in the reaction [34]. PCNA-dependent long-patch
BER was also demonstrated in extracts of Polb-defici-

ent MEF cells, but it appeared to be heavily dependent
on the use of circular DNA substrates [38,41]. During
the PCNA-independent long-patch BER mode, Polb
may be the major DNA polymerase in strand displace-
ment DNA synthesis [40]. However, comparative ana-
lysis of BER in wild-type and Polb null cell extracts
showed the occurrence of long-patch BER, even in the
absence of Polb, suggesting that various DNA poly-
merases provide functional redundancy in long-patch
BER DNA synthesis [38,41].
Various interactions among BER proteins may alter
the choice of BER subpathways. Ape1, when bound to
DNA, interacts with Polb, which also physically inter-
acts with the scaffold protein, XRCC1 [33,50,51].
Poly(ADP-ribose) polymerase (PARP-1), the
enzyme that immediately binds to the incised AP site
and undergoes self-ADP-ribosylation, interacts with
XRCC1 and Polb and affects BER [51,52]. The
involvement of PARP-1 can increase the overall BER
rate, especially by enhancing short-patch BER, by ant-
agonizing the action of Polb, producing a complete
block of long patch BER strand-displacement DNA
synthesis [53]. Long-patch BER reactions are also
well co-ordinated through protein–protein interactions
between PCNA and various BER enzymes, including
Polb, Pold ⁄ e, FEN1 and DNA ligase I [9,54–56]. When
such interactions are disrupted by p21-derived peptide
that binds specifically to PCNA, the mode of AP site
repair was skewed towards short-patch BER, but only
in the presence of Polb [41,57]. Recently, adenomatous

polyposis coli, the tumor suppressor protein, has been
implicated in preventing Polb-mediated strand dis-
placement synthesis by masking the domain of Polb
that interacts with PCNA, thereby decreasing long-
patch BER, but not short-patch BER [58].
An additional variation of BER has been suggested,
as some bifunctional DNA glycosylases are associated
with AP lyase activity that can carry out the cleavage
of AP sites by b-elimination. These reactions generate
3¢ termini that are blocked by the lyase product,
which must be removed by an enzyme, such as Ape1,
to allow repair DNA synthesis (Fig. 2). In this path-
way, the 3¢ repair diesterase activity of Ape1 plays an
important role [59], as it also does in the excision of 3¢
phosphoglycolate esters generated by ionizing radiation
or chemical oxidation [29,60]. More recently, human
polynucleotide kinase has been implicated in the repair
of 3¢ phosphate damage, and its interaction with other
BER proteins, including XRCC1, Polb and DNA
ligase III, has been shown [61].
In general, long-patch BER has been considered to
be a minor pathway relative to the predominant short-
patch BER. However, several in vitro and in vivo stud-
ies suggest a significant contribution of the long-patch
BER mode in some circumstances, particularly in the
repair of regular AP sites or of the damaged base
lesions that become AP sites by the action of mono-
functional DNA glycosylases [39,41,62,63]. As meas-
ured by an in vivo assay using a plasmid containing a
single AP site in the stop codon of the gene encoding

enhanced green fluorescent protein, > 80% of the
repair accompanying the reversion of the stop codon
occurred by long-patch BER [63]. This result is consis-
tent with a previous observation that 70–80% of
uracil-initiated BER was mediated by long-patch BER,
when examined by utilizing a circular DNA substrate
and cell-free extracts of MEF cells [41].
The detailed mechanism that governs the selection
between the short- or long-patch BER modes remains
a major unknown. Previously, it has been suggested
that it is the nature of the DNA lesion that determines
the type of DNA glycosylase (monofunctional versus
glycosylase lyase), which, in turn, determines the
selection of the repair pathway [39]. BER, initiated by
bifunctional DNA glycosylases with associated AP
lyase activity, is mainly mediated by the short-patch
pathway because the resulting BER intermediate,
containing a single nucleotide gap bracketed by a
J S. Sung and B. Demple BER of oxidized abasic sites
FEBS Journal 273 (2006) 1620–1629 ª 2006 The Authors Journal compilation ª 2006 FEBS 1623
3¢-hydroxyl and a 5¢-phosphate, can be readily filled in
by Polb. In contrast, DNA repair, involving a mono-
functional DNA glycosylase that generates an AP site,
may involve both the short- and long-patch BER path-
ways. In this model, the removal of 5¢-dRp, which
appears to be the late-limiting step in short-patch BER
[64], may be critical in determining the mode of BER.
DNA–protein crosslink formation
in the short-patch BER of dL
Chemical methods for the specific generation of dL

lesions within DNA oligonucleotides have been inde-
pendently developed by several laboratories [65–67].
All of these methods involve the photolysis of a stable
precursor and its conversion to dL at a defined site in
synthetic DNA oligonucleotides. These approaches
facilitated the study of the biological fate of this key
oxidative deoxyribose damage in DNA. Initial investi-
gation of dL repair by Escherichia coli endonuclease
III, a bifunctional DNA glycosylase associated with
AP lyase activity, revealed the formation of a sta-
ble DNA–protein crosslink (DPC) with dL, which was
dependent on the lyase active-site lysine residue
involved in b-elimination [19]. Bifunctional DNA gly-
cosylase ⁄ AP lyase enzymes (hOGG1 and hNth1) found
in human cells, can also crosslink to dL [68]. On the
other hand, the E. coli AP endonucleases exonuclease
III and endonuclease IV can efficiently incise dL resi-
dues [68,69]. Consistent with these observations, the
dL-induced mutation frequency measured in vivo was
32-fold elevated in AP endonuclease-deficient E. coli
compared with wild-type bacteria [70].
Human Ape1 protein also incises dL residues rather
efficiently, with a turnover rate (2.3 s
)1
) essentially
identical to that of regular AP sites (2.4 s
)1
), and only
a modest K
m

difference (98 nm for dL versus 21 nm
for AP) [69]. Considering the abundance of Ape1 in
most mammalian cell types, the most probable fate of
dL residues in vivo would be cleavage on the 5¢ side to
yield strand breaks with 5¢-terminal dL-5-phosphate
residues (5¢-dLp). The equivalent 5¢-dRp residue is
effectively processed by the dRp lyase activity of Polb
during short-patch BER. However, reactions of puri-
fied Polb with DNA oligonucleotide substrates con-
taining Ape1-cleaved 5¢-dLp residues led to the
spontaneous formation of covalent crosslinks between
the DNA and the polymerase [71]. The formation of
such DPCs was shown to be dependent on the dRp
lyase active site Lys72 of Polb [71], suggesting that the
respective lysine side chains are involved in nucleophi-
lic attack on the carbonyl carbon of dL, resulting in
the formation of a stable amide bond (Fig. 3B). It has
been shown that bacterial nucleotide excision repair
can incise DNA containing an AP lyase (or peptide)
covalently cross-linked by chemical reduction in an
unbroken DNA [72,73]. Unlike the dL-mediated DPC
formed with an AP lyase on an unbroken DNA, the
DPC formation by Polb trapping to dL occurs at the
DNA strand break generated by Ape1 (compare
Fig. 3B with Fig. 3C). Whether a DPC located at a
DNA strand break can be handled by nucleotide exci-
sion repair remains to be addressed.
In an effort to determine the biological significance
of such crosslink formation, a cell-free extract system
was utilized to react with oligonucleotide DNA con-

taining a site-specific dL residue [57]. Under nonrepair
conditions (no added dNTPs or Mg
2+
), the most pre-
dominant DPC species was found to contain Polb,
because this species was not observed in the reactions
with extracts of Polb null mouse cells. As the dRp
lyase activity of Polb constitutes the major activity for
removing 5¢-dRp residues in mammalian cells [32,44],
the results indicate that DPC formation, specific to the
5¢-dLp lesion, occurs mainly through the abortive
attempt of the dRp lyase activity of Polb to remove
this incised dL lesion. Polb displays strong affinity for
5¢-dRp residues at the incised AP site, while Ape1
recruits Polb to the incised AP site and stimulates its
dRp lyase activity [50,74]. Thus, this enzyme–substrate
specificity may promote the interaction of Polb with a
5¢-dLp lesion at a DNA nick, thereby increasing the
rate of Polb-specific DPC formation. On the other
hand, it has been recently verified that dRp lyase activ-
ity lags behind the polymerase activity in the dual
functions of Pol b, while Ape1 suppresses the poly-
merase activity [75]. In this scenario, Ape1 may modu-
late Polb to pause prior to acting at the 5¢-dLp,
possibly suppressing an abortive attempt to excise the
lesion. Whether interactions between Ape1 and Polb,
or the involvement of other factors, stimulates or
inhibits the covalent trapping of Polb to the 5¢-dLp
residue, must await further analysis.
Use of long-patch BER in the repair

of dL
The major difference found in the sequential enzymatic
steps between short-patch and long-patch BER is the
removal of the incised abasic residue (5¢-dRp). While
the dRp lyase activity of Polb participates in the
processing of this residue, an attempt to remove the
5¢-dLp residue by Polb using the same mechanism
results in trapping of the repair enzyme at the lesion.
In the alternative long-patch BER pathway, removal
of the 5¢-dRp moiety is independent of the Polb dRp
BER of oxidized abasic sites J S. Sung and B. Demple
1624 FEBS Journal 273 (2006) 1620–1629 ª 2006 The Authors Journal compilation ª 2006 FEBS
lyase activity and is mediated mainly by strand dis-
placement DNA synthesis followed by FEN-1 excision.
Therefore, it is not unreasonable to expect that the
Ape1-incised dL residue may be repaired by the long-
patch BER pathway.
Reconstitution of dL-mediated BER conducted with
partial components of long-patch BER, including
Ape1, Polb and FEN-1, revealed that the formation of
dL-mediated DPC was dependent on both Ape1 (for
cleavage) and Polb, but that the amount of this DPC
product was markedly decreased in reactions including
FEN-1 and dNTPs (Fig. 4A). Repair DNA synthesis,
displacing the 5¢-dLp residue by Polb alone, did not
block the DPC formation, indicating that removal of
dL-containing DNA fragment by FEN1 plays a key
role in preventing crosslinking with the DNA substrate
(Fig. 4B). This result suggests that sequential enzymatic
activities in long-patch BER can effectively process the

lesion and avoid dL-mediated DPC formation. This
hypothesis was further supported by the demonstration
of efficient processing of a 5¢-dLp flap oligonucleotide
by FEN-1 [57], consistent with previous observations
showing that the enzyme tolerates a variety of small
modifications of the flap 5¢ terminus [76]. Investigation
of dL-mediated long-patch BER was performed by util-
izing circular DNA with a defined dL residue, incuba-
ted with whole-cell extracts [57]. The repair of dL was
detected in both wild-type and Polb-null MEF cell
extracts, with concomitant reduction of subsequent
crosslinking activity. Analysis of the patch size distribu-
tion associated with BER of site-specific lesions showed
that the single-nucleotide replacement was the predom-
inant repair patch (35% of the total) for a regular AP
site in the Polb-proficient cell extract, but this event
A
B
X=dL
*=
32
P
dL
5'-dL
Polβ
1
2
3
4
C

*
*
*
*
*
Fig. 4. In vitro reconstituted long-patch base excision DNA repair (BER) mediates the repair of 2-deoxyribonolactone (dL) and inhibits the
formation of a dL-mediated DNA–protein crosslink (DPC). (A) A duplex 3¢
32
P-labeled DNA substrate, containing a site-specific dL, was incu-
bated with different combinations of Ape1, DNA polymerase b (Polb) and FEN1 in the presence or absence of a dNTP mix excluding dTTP.
After the incubation, one-half of each reaction mixture was analyzed on a DNA sequencing gel. Ape1 converted the majority of the DNA sub-
strate to the DNA cleavage product, while additional treatments with Polb and FEN1 mediated further processing of the DNA only in the
presence of dNTPs. The generation of the 11-mer is consistent with strand displacement DNA synthesis of seven nucleotides by the poly-
merase, followed by removal of the displaced DNA flap by FEN1. (B) The remainder of each reaction mixture was analyzed by SDS ⁄ PAGE.
The dL-mediated DPCs with Polb are observed with mobilities slower than those of Polb and the free DNA. The generation of DPC was
markedly reduced when the reaction allowed the combined action of repair synthesis by Polb and flap excision by FEN1. (C) Schemes for
the Ape1 incision of DNA at the 5¢ side of the dL lesion (1), the strand displacement DNA synthesis of seven nucleotides by the DNA poly-
merase activity of Polb (2), removal of the 5¢-dLp-containing flap by FEN1, resulting in a nick on DNA (3), and DPC formation via an abortive
attempt to remove the 5¢-dLp residue by the dRp lyase activity of Polb (4). The combined processes of (2) and (3) mediate removal of the
dL-containing oligonucleotide fragment from the DNA substrate and prevent DPC formation with Polb (4). Adapted from a previous publicat-
ion [57].
J S. Sung and B. Demple BER of oxidized abasic sites
FEBS Journal 273 (2006) 1620–1629 ª 2006 The Authors Journal compilation ª 2006 FEBS 1625
was significantly reduced (< 10% of the total) for
repair of the dL substrate. Instead, repair patches of
two or more nucleotides were the predominant mode
for dL with both Polb-proficient and -deficient cell
extracts. It was also confirmed that only the long-patch
BER mode was mostly associated with the complete
repair process, including the final DNA ligation step

[57]. Therefore, at least in mammalian cell extracts, dL
appears to be resistant to repair by short-patch BER,
but effectively and exclusively repaired by long-patch
BER, thereby preventing the formation of deleterious
DPC adducts in DNA.
Concluding remarks
In spite of numerous efforts in defining the biological
and biochemical mechanisms involved in BER, the cel-
lular choice of the specific BER mode remains an
intriguing question. A similar diversity in BER modes
is also found in E. coli [77–79], which indicates that
multiple subpathways of BER are favored by evolution
for defending against various types of nonbulky dam-
age lesions in the genetic material. Our studies of
dL-mediated BER provide at least one clear rationale
for the evolution of long-patch BER to handle a
naturally occurring lesion. While dL residues present
serious problems for cells by mediating stable DPC
formation with Polb, particularly in the course of the
short-patch BER pathway, it appears that the operat-
ion of the long-patch BER pathway substantially
avoids this detrimental consequence. However, under
conditions of extensive oxidative stress, it seems poss-
ible that long-patch BER components may become
limiting because of their participation in the repair of
many other lesions, with the attendant hazards if
short-patch BER increasingly attempts to handle dL
lesions. On the other hand, the induction of proteins
that could modulate the subpathways of BER, as
shown with p21, may alter the outcome of BER oper-

ating on dL [57]. In such circumstances, Ape1-incised
dL residues could remain in the DNA for longer peri-
ods, increasing the opportunity for DPC formation.
Further studies of dL will provide more understanding
the BER switching mechanism that governs the short-
versus long-patch BER distribution under varying
circumstances of damage load and repair enzyme avail-
ability.
Acknowledgements
Work in B. Demple’s laboratory was supported by
NIH grants GM40000 and CA71993. J. S. Sung was
partly supported by Dongguk University Research
Fund. We are grateful to our colleagues, especially Dr
M. S. DeMott, for helpful discussions.
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