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Review
Zebrash as a model system to study DNA damage and repair
De-Sheng Pei
a,b
, Phyllis R. Strauss
b,
a
Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 401122, China
b
Department of Biology, Northeastern University, Boston, MA 02115, USA
a r t i c l e i n f o
Article history:
Received 17 August 2012
Received in revised form19 October 2012
Accepted 23 October 2012
Available online xxx
Keywords:
DNA repair
Base excision repair
Zebrash
Embryological development
p53
a b s t r a c t
Zebrash (Danio rerio) have become a popular vertebrate model to study embryological development,
because of unique advantages not found in other model systems. Zebrash share many gene functions
with other vertebrates including humans, making zebrash a useful system for studying cancer etiology.
However, systematic studies of DNA damage and repair pathways using adult or embryonic zebrash have
not been extensively reported. The zebrash genome contains nearly all the genes involved in different
DNA repair pathways in eukaryotes, including direct reversal (DR), mismatch repair (MMR) nucleotide
excision repair (NER), base excision repair (BER), homologous recombination (HR), non-homologous end
joining (NHEJ) and translesion synthesis (TLS). It also includes the genes of the p53-mediated damage
recognition pathway. Therefore, zebrash provide an ideal model for gaining fundamental insights into
mechanisms of DNA damage and repair, especially during embryological development. This review intro-
duces recent work on different DNA damage and repair studies in zebrash, with special emphasis on the
role of BER in zebrash early embryological development. AP endonuclease 1 (Apex1), a critical protein in
the BER pathway, not only regulates BER but also controls cyclic AMP response binding protein (Creb1),
which itself regulates 25% of eukaryotic coding sequences. In addition, Apex1 indirectly regulates levels
of p53. As these ndings also occur in murine B cells, they illustrate the usefulness of the zebrash system
in elucidating fundamental mechanisms.
2012 Published by Elsevier B.V.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. DNA-repair pathways studied in zebrash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Direct reversal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Nucleotide excision repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Base excision repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.4. Mismatch repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5. Non homologous end joining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.6. Homologous recombination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.7. Translesion synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. The role of BER pathway in zebrash embryological development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Crosstalk between DNA repair and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Concluding remarks: how information from DNA repair could be useful to the zebrash community and why zebrash are an . . . . . . . . . . . . . . . . 00
ideal system for those who study DNA repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conict of interest statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Corresponding author at: Northeastern University, Boston, MA 02215, USA.

Tel.: +1 617 373 3492; fax: +1 617 373 3724.
E-mail addresses: deshengpei@gmail.com(D.-S. Pei), p.strauss@neu.edu
(P.R. Strauss).
1. Introduction
Zebrash (Danio rerio) is a favorite vertebrate model systemfor
developmental and genetic studies because of its initially trans-
parent small body, short reproductive cycle, high reproductive
capacity, andthe ability todoforwardandreverse genetics [15]. In
fact, zebrash could become an ideal model to study DNA damage
0027-5107/$ see front matter 2012 Published by Elsevier B.V.
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and repair pathways. First, zebrash genomic DNA contains ortho-
logues of genes involved in all the DNA repair pathways in higher
eukaryotes (Table 1). Second, it is easy to perform morpholino-
based [6,7] or shRNA knockdown [8,9] studies to directly address
the role of specic DNA damage response genes in each repair
pathway. Third, a new mutagenesis technology involving locus-
specic zinc-nger nucleases (ZFNs) and transcription activator
like effector (TALE) proteins uses the NHEJ pathway to manipulate
the zebrash genome genetically [1015]. ZFNs and TALEs are a
fusion between the cleavage domain of the FokI restriction enzyme
and zinc-nger motifs or transcriptional activators designed to rec-
ognize a specic DNAtarget sequence. They can introduce genomic
lesions at a specic site to stimulate NHEJ-mediated repair of tar-
geted double strand breaks (DSBs) and thus generate mutations in
selectedDNArepair genes. Therefore, the role of specic DNArepair
genes can be addressed directly. Moreover, casper transparent
zebrash mutants are an asset to both toxicology and carcinogene-
sis studies for monitoring damage-induced phenotypes fromlater
development to adult stages [16]. These strategies are helpful to
explore whether selected gene products in different DNA repair
pathways may play additional roles inembryological development.
This reviewfocuses on recent work on DNA damage and repair and
the subsequent DNAdamage response (DDR) in zebrash with spe-
cial emphasis on base excision repair (BER) in early embryological
development.
2. DNA-repair pathways studied in zebrash
Althoughzebrashhave proveduseful intoxicology andembry-
onic development studies for some time, they have not been used
to their fullest potential in studies of DNA damage and repair.
While many zebrash studies focus solely on cytotoxicity or p53-
mediated response to DNA damage, the outcomes are ultimately
the results of specic DNA damage and the related repair path-
ways. Thesepathways includedirect reversal (DR), mismatchrepair
(MMR), nucleotide excision repair (NER), BER, non-homologous
end joining (NHEJ), and homologous recombination (HR) pathway
(Tables 1and2). Note that DSB, where bothstrands of the DNAhelix
are disrupted, are repaired by NHEJ or HR, depending on the nature
of the break and the stage of the cell cycle. Each pathway involves
recognition of the specic damage, damage removal, which may
involve cleavage of one or both strands, resynthesis and ligation of
the repaired strand(s). While the mechanisms in each pathway are
distinct, there is some overlap among pathways.
2.1. Direct reversal
The DR pathway does not involve breakage of the phosphodi-
ester backbone but instead reverses the damage specically and
directly [17]. The prototype enzyme for DR is methyl guanine
methyl transferase (Mgmt), which reverses alkylation of gua-
nine. Although the zebrash genome contains mgmt, no studies
in zebrash have been published at this time. However, a second
system of particular physiological signicance for sh is pho-
toenzymatic repair (PER), which reverses the formation of UV-B
(315280nm) or UV-C (280100nm)-induced adjacent cyclopy-
rimidine dimers (CPD). PER is carried out by a single enzyme,
deoxyribodipyrimidine photolyase, encoded by phr. Notably, PERis
not found in placental mammals, which rely solely on the less ef-
cient NER[18]. PERis activatedby irradiationwithlong wavelength
(300500nm) UVA/visible light [1921]. Fidelity of photo repair
has been evaluated in zebrash embryos exposed to UVB followed
by UVAexposure torepair CPD[19,2224]. Note that oxidative dam-
age resulting fromexposure to ultraviolet light is repaired by base
excision repair (see below) [18,22,23,2528].
2.2. Nucleotide excision repair
NER, found in all organisms including sh [29], excises and
repairs pyrimidine dimers. There are two NER pathways distin-
guished by whether the lesion is on the transcribed strand of an
active gene (TCR) or the rest of the genome (GGR) [30]. There are
no studies at this time that distinguish TCR fromGGR in zebrash,
although the presence of the PER system could conceivably alter
the balance betweenTCRandGGR. Other studies relate primarily to
dose dependence of exposure of whole zebrash or zebrash liver
cells [31,32] toUVandtheresponsetoUVexposureof various genes
involved in NER such as cdkI (cyclin-dependent kinase inhibitor),
xpc, ddb2, p53, gadd45a and cyclinG1 [25,29,3336]. Interestingly,
after exposure to wastewater efuent, adult male zebrash and
zebrash liver cells demonstrated altered xpc and xpa expressions
[37]. Alterations depended on the source of waste water and the
time of year. xpd fromzebrash was cloned and the expression pat-
tern in different tissues and different stages was investigated [38].
While xpd/ercc2 was expressed in all developmental stages includ-
ing unfertilized eggs, [38], expression of xpa could not be detected
by western blot analysis in early stage embryos prior to hatching,
even after UV irradiation [24,39,40].
Exposure to the potent synthetic estrogen, 17alpha-
ethinylestradiol (EE2) caused signicant decreases in several
hepatic NER genes, indicating potential decreased NER capacity
and presumably increased cancer risk. In fact, exposure of cultured
zebrash liver cells to physiologically relevant concentrations
of EE2 reduced the ability to repair a damaged reporter plasmid
[35].
2.3. Base excision repair
BER recognizes non-bulky lesions such as 8-oxoguanine (8-
oxoG) and the presence of uracil or abasic (AP sites) (Fig. 1). The
lesion is recognized by a glycosylase that removes the inappropri-
ate base without cleaving the backbone and generates an AP site
(Fig. 1, Reaction 1). The AP site is then cleaved 5

to the deoxyri-
bosephosphate by AP endonuclease 1 (Apex1), generating the free
3

hydroxyl group required for the repair polymerase (Fig. 1, Reac-

tion 2). The dangling dRP is removed by DNA polymerase (PolB),
which lls the gap (Fig. 1, Reactions 3a and 4a). Some glycosylases
not only remove the offending base but also nick the AP site on
the 3

side, leaving a blocking lesion for PolB. In that case, Apex1 is

required to trim the dRP, which results in a single nucleotide gap
that is lled by PolB after which a ligase seals the nick (Fig. 1, Reac-
tion 5a). On occasion, chemical modication prevents removal of
the dRP. Under those conditions, PCNA and one of the replicative
polymerases (or possibly PolB) displaces the downstream strand
and inserts up to 5 nucleotides (Fig. 1, Reaction 3b). The displaced
strand is cleaved by ap endonuclease1 (Fen1) (Fig. 1, Reaction 4b)
and a ligase seals the nick (Fig. 1, Reaction 5b). Accessory proteins
include Xrcc1 and Parp. Biochemical studies of zebrash enzymes
involved in BER include PolB [41] and Apex1 [26]. Like Xpa, PolB
is missing in early stage zebrash embryos [28] so that before 13h
post fertilization (hpf), BER in early stage embryos relies on one or
more aphidicolin-sensitive DNA polymerase(s) [28]. Detailed stud-
ies of pathway regulation as studied in zebrash and in mice are
presented in Section 3 below.
Toxicity studies of agents that result in oxidative damage to
DNA that are likely to be repaired by BER in zebrash embryos
or adults include those involving chronic oxidation from expo-
sure to copper [33], and the pyrethroid insecticide cypermethrin
[4244]. Chronic exposure to the former resulted in decreased
apex1 expression compared to control, while exposure to cyper-
methrin down-regulated ogg1 gene expression, induced oxidative
stress and upregulated antioxidant proteins (Cu/Zn-dependent
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Table 1
The major genes of different DNA repair pathways in zebrash.
Types of DNA repair Major genes Accession ID Protein size (aa)
Direct reversal mgmt XP 684479.2 187
phr NP 957358.1 516
Base excision repair ung NP 957268.1 291
ogg1 NP 001074145.1 268
apex1 (S, L)
a
NP 998586.1 310
parp1 (S) NP 001038407.1 1013
polb (S) NP 001003879 337
xrcc1 (S) NP 001003988.1 615
lig3 (S) NP 001025345.1 752
pol (L) NP 001034899.1 1105
pol (L) XP 001920422.1 2288
fen1 (L) NP 942115 330
lig1(L) XP 685041.2 1058
Mismatch repair msh2 NP 998689.1 936
msh3 NP 001103184.1 1083
msh6 NP 878280.1 1369
mlh1 NP 956953.1 724
pms2 XP 693648.3 851
exo1 NP 998634.1 807
pol NP 001034899.1 1105
Nucleotide excision repair RNA polII (TCR)
b
XP 682682.1 1972
csa/ercc8 (TCR) NP 001005984.1 400
csb/ercc6 (TCR) XP 688972.2 1390
rpa (TCR, GGR)
c
NP 956105.2 601
xpa (TCR, GGR) NP 956765.1 549
xpg (TCR, GGR) NP 001014337.1 249
ercc1 (TCR, GGR) NP 001096608.1 342
xpc NP 001038675.1 879
xpe NP 956920.1 897
hr23b NP 956858.1 382
ddb2 NP 001076530 497
Nonhomologous end joining ku70 NP 956198.1 409
ku80 NP 001017360.1 727
DNA-PK/prkdc XP 001919588.1 4119
xrcc4 NP 957080.1 357
lig4 NP 001096593.1 909
artemis NP 001038566.1 639
Homologous recombination atm XP 001334561.2 323
rad51 AAH62849.1 338
rad52 NP 001019622.1 409
rad54 NP 957438.1 738
pol NP 001034899.1 1105
pol XP 001920422.1 2288
lig1 XP 685041.2 1058
NHEJ and HR rad50 XP 696859.3 1332
mre11 NP 001001407.1 619
nbn NP 001014819.1 818
Translesion synthesis poleta NP 001035337.1 743
poliota NP 001017834.1 710
polkappa XP 691219.4 902
pollambda NP 998408.1 566
polmu NP 956542.1 507
polnu NP 001093496.1 1146
rev1 NP 001116772.1 1268
polzeta XP 002665692.2 1615
p53-Mediated Surveillance p53 NP 571402.1 373
mdm2 NP 571439.2 475
atm XP 002664603.2 2451
atr XP 696163.4 2643
p21 XP 003200962 170
survivin (birc5a) NP 919378.1 142
rad1 NP 957192.1 279
rad9 XP 692358.2 402
hus1 NP 001082965.1 284
baxa NP 571637.1 192
noxa (pmaip1) NP 001038939 45
puma (bbc3) NP 001038937.1 181
bcl2 NP 001025424.1 228
bcl-xl NP 571882.1 238
casp 2 NP 001036160.1 435
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Table 1 (Continued)
Types of DNA repair Major genes Accession ID Protein size (aa)
casp 3a NP 571952.1 282
casp 6 NP 001018333.1 298
casp 7 NP 001018443.1 316
nur77 (nr4a1) NP 001002173.1 574
p73 NP 899183.1 640
a
S short patch, L long patch.
b
TCR transcription coupled repair.
c
GGR global genomic repair.
superoxide dismutase, Mn-dependent superoxide dismutase, cata-
lase, and glutathione peroxidase), resulting in apoptosis with
caspase activation in zebrash embryos.
2.4. Mismatch repair
Like BER, MMR repairs non-bulky lesions in DNA. However,
the use of the word lesion is a misnomer, because the pathway
is limited to recognition and repair of mispaired but undamaged
bases. For that reason it plays a key role in maintaining genomic
stability and suppressing homologous recombination. Defects in
MMR are associated with predisposition to colon cancer including
Hereditary Non-Polyposis Colorectal Cancer (HNPCC), resistance
to certain chemotherapeutic agents, abnormalities in meiosis and
male sterility. These outcomes have all been studied in zebrash
systems. Some of the zebrash genes involved in MMR, notably
the damage recognition proteins Msh6, Msh2, and Mlh1 [45,46],
have been cloned and both temporal and spatial distribution
have been followed [45,46]. However, zebrash mlh3, replication
Table 2
Recent studies on DNA repair pathways in zebrash.
DNA repair pathway Topic Genes involved Reference
DR UV-damaged-DNA binding activity in zebrash [24]
Photobiological effects of UV pho [22]
UVA-induced photo recovery pho [23]
BER Apex1 affects heart,brain and blood development apex1 [26]
Copper exposure in zebrash apex1 [33]
BER in early zebrash development apex1, polb [27]
A novel regulatory circuit in BER pathway apex1, creb1, polb [28]
Overexpression of Polb in E. coli polb [41]
Cypermethrin exposure in zebrash ogg1, p53 [44]
NER 17-Ethinylestradiol affects NER genes expression xpc, xpd, xpa, xpf [34]
UVC and oxidative damage in cultured sh cells [25]
17 -Ethinylestradiol hinders NER in zebrash liver cells xpc, xpa [35]
p53-dependent NER pathway in zebrash p53 [29]
Time-course expression of DNA repair-related genes by UVB p53, xpc, ddb2, gadd45a, cyclinG1 [33]
Wastewater treatment alters NER in zebrash xpc, xpa [37]
Cloning of xpd in zebrash xpd [38]
1,10-phenanthroline (OP) stimulated UV damaged DNA binding proteins vitellogenin [39]
UVR exposure in sh embryos [40]
EE2 modulates p53 but not NER pathways p53, p21, gadd45a [36]
MMR MMR-dependent chromosomal instability fromalkylation damage msh6 [53]
Mlh1 deciency for zebrash male sterility mlh1 [48]
Completion of meiosis lack of mlh1 in zebrash mlh1 [49]
MMR deciency does not enhance ENU mutagenesis msh6 [51]
Mutations in MMR develop tumors mlh1, mlh2, mlh6 [50]
Cadmiumdown-regulates msh6 msh6 [68]
Cloning of msh2 in zebrash msh2 [46]
Cloning of msh6 in zebrash msh6 [45]
Zebrash: chiasmata and interference mlh1 [47]
NHEJ SB transpositional recombination by NHEJ pathway dna-pk [69]
Ionizing radiation-induced DNA damage. xrcc5/ku80 [54]
Ku70 protects nervous systemfromradiation damage xrcc6/ku70 [55]
The role of NHEJ in transgene concatemer formation [57]
ZFN targeted disruption in zebrash [11]
ZFN minireview [10]
ZFN targeted disruption in zebrash [14]
ZFN as gene therapy agents [70]
NHEJ involved in LINE retrotransposition z2-2, ku70, art, ligIV [56]
Rapid mutation of zebrash gene by ZFN OPEN [13]
Targeted mutagenesis using customized ZFN [12]
Improved somatic mutagenesis in zebrash using TALENs [15]
HR Chi-stimulated HR and its application in zebrash [71]
HR and NHEJ in zebrash [59], [58]
Use of RecA fusion proteins for zebrash genomic modications reca [62]
Rad52 and RPA mediated mutagenesis rad52, rpa [61]
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Fig. 1. DNA base excision repair pathway (modied fromFortier et al. [23]). DNA base excision repair pathway. The step number is indicated at the left in Arabic numerals.
Steps 3a, 4a, and 5a occur in short patch (single-nucleotide) repair. Steps 3b, 4b, and 5b occur in long patch repair (insertion of two to six additional nucleotides). Step 3b
uses the replicative DNA polymerase-, -, and -, in the presence of ap endonuclease 1 (FEN1) and PCNA (not shown).
factor c (rfc) and pms2 remain to be explored. Expression of msh6
relative to msh2 and the tissue distribution of msh2 varied with
the stage of development [45] until the relative levels stabilized
at 120 hpf [46]. msh2 tended to be localized in the brain, eyes,
telencephalon, and the fourth ventricle at 12 days post fertilization
(dpf)-48dpf embryos [46]. Onthe other hand, Mlh1foci detectedby
immunouorescence tended to be in the distal regions of zebrash
synaptonemal complexes (SCs) [47].
MMR is critical for sperm maturation. Male zebrash lacking
the Mlh1 protein were largely infertile. Since spermatogenesis
arrested at metaphase I of meiosis and cells died by apoptosis,
later germ cell stages were absent [48]. Nevertheless, spermato-
genesis was still completed by a limited number of germ cells
[49]. Although no studies of mutation in specic genes were per-
formed, eggs fertilized with surviving spermproduced malformed
embryos.
Although sh that are homozygous mutant for the MMR genes
mlh1, msh2, and msh6 developed neoplasms particularly neuro-
bromas and malignant peripheral nerve sheath tumors of the eye
and abdomen [50], mutagenesis induced by exogenous agents was
not affected by loss of MMR gene function. For example, ethyl-
nitrosourea (ENU) exposure is a standard protocol for generating
germ line mutations in zebrash. Although one might expect that
successful mutagenesis would be enhanced by loss of MMR, de-
ciency in the MMR gene msh6 did not enhance ENUmutagenesis in
the zebrashgermline [51]. MMR alsocontributedtogrowtharrest
in response to DNA damage by alkylating agents [52]. Although the
primary response toalkylationdamage occurs via BER, invivo expo-
sure of zebrash eggs to alkylating agents resulted in chromosomal
instability and cell death due to stalled replication forks normally
processed by MMR [53].
2.5. Non homologous end joining
NHEJ is a pathway that repairs double-strand breaks in DNA
that arise from exposure to UV, ionizing radiation (IR) or extreme
damage from alkylating agents otherwise repaired by BER. This
repair pathway is relatively inaccurate. The DNA ends are recog-
nized by Ku70 and Ku80 that recruit the proteins that will perform
end-rejoining without requiring a homologous template. ku80 and
ku70 of the zebrash NHEJ pathway have been cloned [54,55].
Ku80 promoted survival of irradiated cells during embryogenesis.
Radiation-induced apoptosis in the Ku80 knockdown zebrash is
suppressed by p53 knockdown, indicating that apoptosis result-
ing from loss of Ku80 is p53 dependent [54]. Ku70 protein played
a crucial role in protecting the developing nervous system from
radiation-induced DNA damage during embryogenesis [54]. NHEJ
is involved in retrotransposition of long interspersed elements
(LINEs) from both zebrash and mammalian sources. The use of
zebrashLINEs enabledresearchers todemonstratetheimportance
of Ku70 and Artemis in chicken B lymphocyte lines defective for
these genes [56]. Furthermore, microinjection of linearized DNA
encoding green uorescent protein into zebrash embryos was fol-
lowed directly by uorescence microscopy after which the DNA
was recovered and sequenced. Fromthese data Dai et al. concluded
that NHEJ is the principal mechanism of exogenous gene integra-
tion in zebrash [57]. Recently, Liu et al., developed a visual-plus
quantitativeanalysis systems tostudyDNADSBrepairs inzebrash,
which also showed that NHEJ was predominant among the three
DSB repair pathways (NHEJ, HR and SSA) in zebrash embryos [58].
NHEJ is the underlying mechanism for generating targeted
mutations by new, highly efcient methods known as ZFNs
(locus specic zing-nger nucleases) or TALENs (transcription
Please cite this article in press as: D.-S. Pei, P.R. Strauss, Zebrash as a model systemto study DNA damage and repair, Mutat. Res.: Fundam. Mol.
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activator-like effector nucleases) in zebrash and other eukary-
otes [15]. The methodology is relatively simple, leads to indel
(insertion/deletion) formation. Some of the genes that have been
targeted with this methodology to date include kdr [14], golden
and ntl [10,11], transferring receptor 2 (tfr2), dopamine trans-
porter (slc6a3), telomerase (tert), hypoxia inducible factor 1a alpha
(hif1aalpha), gridlock (hey2) [12,13], bmi1, Ikzf1, phf6(1), phf6(2),
MyoD, and Jak3 [15].
2.6. Homologous recombination
HR is the accurate, templated repair of DSBs in the late S-G2
phase of the cell cycle. First a single-stranded region of DNA for
strand invasion is generated, then a Holliday junction is formed
and nally DNA synthesis is initiated using the intact strand as
the template. HR has been reported in intact zebrash embryos
at early developmental stages [59] and in zebrash ES cell cul-
tures [60]. Several attempts to use HR to promote gene targeting in
zebrash have not met with success [61,62], although one recent
report showed that knockdown of Rad51 decreased HR ability in
zebrashembryos [58]. Muchremains tobestudiedinthezebrash
HR system.
2.7. Translesion synthesis
The translesion polymerases enable the dividing cell to synthe-
size past a mispair or a lesion during replication and/or repair. DNA
polymerase eta was the rst to be identied, as it is capable of
bypassing thymine dimer CPDs. Unlike the replicative DNA poly-
merases, , and , these polymerases exhibit low delity when
copying undamaged DNA [6367]. Some like Rev1, however, insert
a correct base (a C residue) opposite a template G but are inaccu-
rate when synthesizing opposite an AP site or certain other minor
groove adducts. DNApolymerase lambda and DNApolymerase mu,
two additional translesion synthesis polymerases found in other
vertebrates, have not yet been identied in the zebrash genome.
There is no published work on the translesion polymerases in
zebrashat this time. Giventherapidityof DNAsynthesis intherst
hours after zebrash fertilization, it seems likely that the transle-
sion polymerases could play an important role [28].
3. The role of BER pathway in zebrash embryological
development
The role of the BER pathway in DNA repair (Fig. 1) is reviewed
in the preceding section. In zebrash unfertilized eggs and early
stage embryos are able to perform at least the rst three BER
steps [26]. However, BER in early stage embryos (before 12hpf) has
several unexpected features consistent with rapid cellular prolifer-
ation (15min/cycle for the rst 10 division cycles). In early stage
embryos, aphidicolin-sensitive DNA polymerase(s) replace PolB,
which is absent, while the presence of backup Mg
2+
-dependent
endonuclease activity means that Apex1 has assistance in case of
overwhelming oxidative damage. When the embryo hatches from
the chorionic membrane and encounters normal oxidative stress,
it switches to normal adult BER [27].
Apex1 is a crucial participant in BER. The knockout of Apex1
in mice is lethal, and no viable null cell line has been created,
indicating that Apex1 is critical for development. Apex1 is highly
conserved in different species and zebrash. Zebrash Apex1
shares the same enzymatic properties as its human orthologue. In
zebrash, Apex1proteinhas 78%homology (64%identity) withthat
of the human protein. Full knockdown of Apex1 in early zebrash
embryos leads to embryonic failure at the midblastula transition
(MBT) stage. The MBT is the stage where cell division slows from
15min/cycle, cells become motile, zygotic transcription is fully
turned on, and spatial differentiation begins. Partial knockdown
of Apex1 causes abnormalities in eyes, notochord, brain, blood
cells and heart [26]. The failure of the heart to develop normally
explains the basis for inability of mouse embryos to develop after
Apex1 knockout: the heart forms early in development in mice and
when heart development fails to proceed normally the embryo is
resorbed efciently. The advantage of the zebrash system is that
zebrash embryos can continue developing for up to seven days
with a non-functional heart. Both western blot and immunohisto-
chemistry using anti-Apex1 antibody showthat Apex1 is abundant
in unfertilized eggs and throughout development (Fig. 2) [26].
The use of zebrash made it possible to discover that Apex1
plays a critical and previously unknown role in cell physiology.
These studies could not have been performed in mammals because
of the developmental requirements described above. Apex1 reg-
ulates the protein level of the crucial transcription factor Creb1
[28]. Consequently, Apex1 regulates the levels of many other Creb-
dependent proteins including PolB, the next protein in the BER
pathway. After knockdown of Apex1, embryos fail to synthesize
Creb1proteinandits bindingpartners, Creb1bindingprotein(CBP),
Creb1 modulating protein (Crem) and Targets of Creb1 (Torcs 1 and
3). Similar effects are seeninprimary cultures of mouse Bcells from
Apex1mice when remaining Apex1 is inhibited by CRT0044876.
Finally, these results reinforce the requirement for endonuclease
activity rather than any redox activity for successful embryogene-
sis and cell survival because rescue requires microinjection of the
gene for the endonuclease competent protein.
4. Crosstalk between DNA repair and apoptosis
Generation of reactive oxygen/nitrogen species and subsequent
oxidative damage to DNAare ongoing processes inevery cell. These
events are distinct fromdouble strand breaks unless the breaks are
very closely spaced or the damage is overwhelming. Nevertheless,
oxidative damage requires repair to maintaingenome integrity and
stability. DNA damage activates checkpoint mechanisms to arrest
the cell cycle in order to allow time to repair the DNA damage. If
the damage is too severe for efcient repair, the cell will be induced
to undergo apoptosis, necrosis, autophagy, paraptosis, or possibly
mitotic catastrophe. The tumor suppressor protein p53 provides
one important signaling mechanism between DNA damage and
apoptosis [72]. Loss or suppression of p53 or disabling mutations
in p53 are associated with a variety of tumors [7378]. Despite
the better understood importance for apoptosis, the p53 family
serves broader and more complex functions than simply reducing
the incidence of cancer. It is involved ina number of non-neoplastic
processes including reproduction, metabolism, development, drug
and radiation toxicity and aging [76,79,80].
Zebrash p53, which is structurally and functionally conserved
with mammalian p53 [81], serves as a key mediator of apoptosis
[76] especially for unrepaired double strand breaks. Most of the
studies involving p53 in zebrash have involved a link to apopto-
sis engendered by either UV or IR radiation, both of which generate
DSBs [76]. Inthat situationp53 mediates apoptosis througha linear
pathway involving Bax transactivation, Bax translocation fromthe
cytosol to membranes, cytochrome c release from mitochondria,
and caspase-9 activation, followed by the activation of caspase-
3, -6, and -7. p53-mediated apoptosis can be blocked at multiple
checkpoints: by inhibiting p53 activity directly, by Bcl-2 family
members regulating mitochondrial function, by E1B 19K blocking
caspase-9 activation, and by caspase inhibitors [82].
Regulation of p53 transcription is complex and not entirely
understood [79]. Among the modulators in zebrash are Mdm2
[83] and 113p53, an alternative splicing p53 isoform [84].
Instead of acting as a dominant negative for p53 [84], 113p53
Please cite this article in press as: D.-S. Pei, P.R. Strauss, Zebrash as a model systemto study DNA damage and repair, Mutat. Res.: Fundam. Mol.
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Fig. 2. Analysis of the protein expression level of Apex1 at different developmental stages in zebrash by confocal microscopy. (A and B) Apex1, a maternal protein, is stored
in the unfertilized egg, as shown by uorescence microscopy (A, FITC-labeled Apex1) and confocal microscopy (B, the merged picture with FITC-labeled Apex1 and propidium
iodide-stained nuclei); (C) 4-cell stage embryo with lateral view (up) and top view (lower right corner); (D) 64-cell stage with lateral view (up) and top view (lower right
corner); (E and F) sphere stage (E) and 50% epiboly stage (F) with the animal pole to the top; (G and H) 10 somite stage (G) and 1 dpf stage (H) with dorsal to the top and
anterior to the left; (I and J) confocal microscopy of a 64-cell stage embryo (J is the enlarged view of the square region in panel I.) The diploid nuclei are clearly visible with
red color. (K and L) The 3.2kb upstreamsequence preceding the ATG start codon on the Apex promoter in zebrash was amplied and inserted into pEGFP-N3 to construct
the pApexp3k-GFP plasmid. Constructs (2nl, 50ng/l) were micro-injected into 2cell stage embryos and GFP expression was examined at 1dpf by.
differentially modulated p53 target gene expression to antago-
nize p53 apoptotic activity after zebrash embryos were exposed
to foreign DNA with broken ends or IR that generated DSBs
[81,84]. Overexpression of 113p53 enhanced p53-dependent
up-regulation of p21 and mdm2, but inhibited p53-dependent up-
regulation of the proapoptotic gene bax [76,84].
Inzebrashacommonmethodologyfor knockingdownselected
proteins in early embryogenesis is the microinjection of mor-
pholino oligonucleotides (MO) designed to target a selected mRNA
[6,85,86]. Unlike inhibitory RNA transfected into mammalian cells,
theMO neednot beprocessedbut, instead, binds directlytoits com-
plementary sequence to inhibit either splicing or translation. Since
MO are stable and non-degradable and interact with target mRNA
in a stoichiometric fashion, effects of knockdown can frequently be
detected for up to seven dpf. As with any knockdown technology,
nonspecic, off target effects occur sothat theknockdownpheno-
type needs to be conrmed by rescue with the appropriate, coding
mRNA. Of interest here is that off target effects can frequently
be corrected by knocking down p53 along with the gene of interest
[87] or performing the knockdown in p53 mutant (p53
M214K/M214K
)
sh [88]. In this line, sh fail to undergo DNA damage-dependent
apoptosis after -irradiation, fail to upregulate p21 after UV irra-
diation and do not arrest at the G1/S checkpoint [89]. Given that
microinjection of DNA with broken ends up-regulates p53 [81],
off-target effects involving activation of p53 should not come as
a surprise.
Our recent study showed that loss of Apex1, one of major pro-
teins in BER pathway, causes selective DNA damage and activates
the p53 pathway (Pei&Strauss, unpublished data). Expression of
p21, mdm2 and 113p53 increased in Apex1 knock down embryos.
The morphants had smaller heads and eyes and abnormal brains.
The morphant phenotype was rescued with mRNA for human
wild-type APEX1 [26]. More important, a similar morphant phe-
notype was seen in p53 mutant (p53
M214K/M214K
) sh and in wild
type sh in which both p53 and Apex1 had been knocked down
(Pei&Strauss, unpublished data). Clearly, Apex1 deciency acti-
vated a p53-dependent pathway but the brain and neural effects
and the loss of Creb1 resulting fromApex1 loss were independent
of p53-mediated apoptosis. On the basis of these results, we pro-
pose that Apex1 not only regulates Creb1 but also that Apex1 be
included as a regulator of p53.
In addition, one might also ask whether the ends of the mRNAin
the double strand mRNA/MO complex might be digested away so
that the resulting double strand fragment is recognized as a double
strand break intermediate. The complex could act as a poison for
the components of DSBR, since the morpholino-containing strand
cannot be degradedreadily. Hence, it wouldtrigger a p53-mediated
apoptotic response
Please cite this article in press as: D.-S. Pei, P.R. Strauss, Zebrash as a model systemto study DNA damage and repair, Mutat. Res.: Fundam. Mol.
Mech. Mutagen. (2012), http://dx.doi.org/10.1016/j.mrfmmm.2012.10.003
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5. Concluding remarks: howinformation fromDNA repair
could be useful to the zebrash community and why
zebrash are an ideal systemfor those who study DNA
repair
Zebrash (D. rerio) provide a bona de vertebrate model sys-
tem for studying DNA damage and its repair. In turn, results from
DNA repair studies are bound to yield a wealth of surprises for
embryologists using the zebrash system. Zebrash have the genes
for most, if not all, the enzymes in various mammalian repair
pathways including direct reversal, mismatch repair, nucleotide
excision repair, base excision repair, homologous recombination,
non-homologous end joining and translesion synthesis. Some
participants such as Xpa and PolB are missing in very early devel-
opment but come on line by the time the embryo hatches fromthe
chorion and encounters the environment in which it will spend
the rest of its life. Other components such as Msh6 and Msh2
vary in their relative amounts or locations as embryogenesis pro-
ceeds until hatching when both levels and locations stabilize. In the
case of PolB, which participates in base excision repair, the lack in
very early embryogenesis is offset by anaphidicolin-sensitive poly-
merase and the embryo retains the full ability to repair oxidative
DNAdamage. The lack of these important components at that stage
could be universal in that there is little information in mammalian
systems as to precisely how DNA is repaired in early embryos.
Some proteins such as Apex1 that participates in base excision
repair play dual roles. Apex1 regulates protein levels of the impor-
tant transcription factor Creb1 which itself regulates transcription
of about 25% of structural genes in the eukaryotic genome. Con-
sequently, Apex1 regulates many other Creb-dependent proteins
including PolB. At this time the mechanism by which Apex1 reg-
ulates Creb1 protein levels is not clear, although the requirement
for Apex1s endonuclease activity hints at a lesion-processing step.
Other proteins involved in DNA repair may play equally complex
and important roles, which remain to be explored. Zebrash are an
ideal systemfor these studies.
Conict of interest statement
The authors declare that there is no actual or potential conict
of interest.
Acknowledgments
The authors are grateful for support from the G. Harold and Y.
Leila Mathers Foundation and fromNortheastern University.
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