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
Trang 1Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA
Jung-Suk Sung1and Bruce Demple2
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 O6-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: bdemple@hsph.harvard.edu
(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-dL-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.
Trang 2reactive 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 unphoto-damaged 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 O2 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
OPO3
OH
AP site
Spontaneous Base Loss Removal of Bases
by DNA Glycosylases
OPO3
OPO3
A
O
2-Deoxyribonolactone
O
OPO3
OPO3
OPO3
5-methylene-2-furanone
N
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).
Trang 3The 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 Pol δ/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
OPO3
OPO3
2-OH
H 2 N
OH
OPO3
H
HN
+
β-elimination
A
K72
5'-dRP lyase
K72
Polβ
OPO3
2-OPO3
2-Polβ
O
OPO3
OPO3
O HN B
H 2 N K72
K72
OPO32- OPO3
2-O
OPO3
O
H 2 N
OH
OPO3
O HN
AP Lyase
C
OPO3
2-OPO3
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.
Trang 4that 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 dependPolb-defici-ent
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-long-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
Trang 53¢-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 Km 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 Mg2+), 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 activactiv-ity in the dual functions of Polb, 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
Trang 6lyase 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
β
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].
Trang 7was 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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