Zebraﬁsh as a Model System to Study DNA Damage and Repair

8/11/2019 Zebrafish as a Model System to Study DNA Damage and Repair

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Please cite this article in pressas: D.-S. Pei, P.R. Strauss, Zebrafish as a model system to study DNA damage and repair, Mutat. Res.: Fundam. Mol.

Mech. Mutagen. (2012), http://dx.doi.org/10.1016/j.mrfmmm.2012.10.003

ARTICLE IN PRESSGModel

MUT-11229; No.of Pages9

2 D.-S.Pei, P.R. Strauss / Mutation Researchxxx (2012) xxxxxx

and repair pathways. First, zebrafish 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 specific DNA damage response genes in each repair

pathway. Third, a new mutagenesis technology involving locus-

specific zinc-finger nucleases (ZFNs) and transcription activator

like effector (TALE) proteins uses the NHEJ pathway to manipulate

the zebrafish genome genetically [1015]. ZFNs and TALEs are a

fusion between the cleavage domain of theFokIrestriction enzyme

and zinc-finger motifs or transcriptionalactivators designed to rec-

ognize a specific DNAtarget sequence. They can introduce genomic

lesions at a specific site to stimulate NHEJ-mediated repair of tar-

geted double strand breaks (DSBs) and thus generate mutations in

selected DNArepair genes.Therefore,the role of specific DNArepair

genes can be addressed directly. Moreover, casper transparent

zebrafish mutants are an asset to both toxicology and carcinogene-

sis studies for monitoring damage-induced phenotypes from later

development to adult stages [16]. These strategies are helpful to

explore whether selected gene products in different DNA repair

pathways may playadditional rolesin embryological development.

This review focuses on recent work on DNA damage and repair and

thesubsequent DNAdamage response (DDR) in zebrafish with spe-

cial emphasis on base excision repair (BER) in early embryological

development.

2. DNA-repair pathways studied in zebrafish

Although zebrafish have proved usefulin toxicologyand embry-

onic development studies for some time, they have not been used

to their fullest potential in studies of DNA damage and repair.

While many zebrafish studies focus solely on cytotoxicity or p53-

mediated response to DNA damage, the outcomes are ultimately

the results of specific DNA damage and the related repair path-

ways.These pathwaysinclude direct reversal (DR),mismatchrepair

(MMR), nucleotide excision repair (NER), BER, non-homologous

end joining (NHEJ), and homologous recombination (HR) pathway

(Tables1and2). Note that DSB, where both strands of theDNA helix

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 specific 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 specifically and

directly [17]. The prototype enzyme for DR is methyl guanine

methyl transferase (Mgmt), which reverses alkylation of gua-nine. Although the zebrafish genome contains mgmt, no studies

in zebrafish have been published at this time. However, a second

system of particular physiological significance for fish 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 byphr. Notably, PERis

not found in placental mammals, which rely solely on the less effi-

cientNER[18]. PERis activatedby irradiation with long wavelength

(300500nm) UVA/visible light [1921]. Fidelity of photo repair

has been evaluated in zebrafish embryos exposed to UVB followed

byUVA exposure to repairCPD [19,2224]. Notethat oxidativedam-

age resulting from exposure 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 fish [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 from GGR in zebrafish,

although the presence of the PER system could conceivably alter

thebalance between TCRand GGR. Other studies relate primarily to

dose dependence of exposure of whole zebrafish or zebrafish liver

cells [31,32]toUV andthe responseto UV exposureof 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 effluent, adult male zebrafish and

zebrafish liver cells demonstrated altered xpcandxpa expressions

[37]. Alterations depended on the source ofwaste water and the

time of year.xpd from zebrafish wasclonedand theexpression pat-

tern in different tissues and different stages was investigated [38].

Whilexpd/ercc2 was expressed in all developmental stages includ-

ing unfertilized eggs, [38], expression ofxpa could not be detected

by western blot analysis in early stage embryos prior t o hatching,

even after UV irradiation [24,39,40].

Exposure to the potent synthetic estrogen, 17alpha-

ethinylestradiol (EE2) caused significant decreases in several

hepatic NER genes, indicating potential decreased NER capacity

and presumably increased cancer risk. In fact, exposure of cultured

zebrafish 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 free3 hydroxyl group required for the repair polymerase (Fig. 1, Reac-

tion 2). The dangling dRP is removed by DNA polymerase (PolB),

which fills 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 filled by PolB after which a ligase seals the nick (Fig. 1, Reac-

tion 5a). On occasion, chemical modification 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 flap 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 zebrafish enzymesinvolved in BER include PolB [41] and Apex1 [26]. Like Xpa, PolB

is missing in early stage zebrafish embryos [28] so that before 13h

post fertilization (hpf), BER in early stage embryos relies on one or

more aphidicolin-sensitive DNApolymerase(s) [28]. Detailed stud-

ies of pathway regulation as studied in zebrafish 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 zebrafish 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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D.-S. Pei,P.R. Strauss / Mutation Researchxxx (2012) xxxxxx 3

Table 1

The major genesof differentDNA repair pathways in zebrafish.

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 RNApolII (TCR)b XP 682682.1 1972

csa/ercc8 (TCR) NP 001005984.1 400

csb/ercc6 (TCR) XP 688972.2 1390rpa (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 338rad52 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

casp2 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 longpatch.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 zebrafish 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

MMRare 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 zebrafish

systems. Some of the zebrafish 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, zebrafish mlh3, replication

Table 2

Recent studies on DNA repair pathwaysin zebrafish.

DNA repair pathway Topic Genes involved Reference

DR UV-damaged-DNA binding activity in zebrafish [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 zebrafish apex1 [33]

BER in early zebrafish 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 zebrafish ogg1, p53 [44]

NER 17-Ethinylestradiol affects NER genes expression xpc, xpd, xpa, xpf [34]UVC and oxidativedamage in cultured fish cells [25]

17-Ethinylestradiol hinders NER in zebrafish liver cells xpc, xpa [35]p53-dependent NER pathway in zebrafish p53 [29]

Time-course expression of DNA repair-related genes by UVB p53, xpc, ddb2, gadd45a, cyclinG1 [33]

Wastewater treatment alters NER in zebrafish xpc,xpa [37]

Cloning ofxpd in zebrafish xpd [38]

1,10-phenanthroline (OP) stimulated UV damaged DNA binding proteins vitellogenin [39]

UVR exposure in fish embryos [40]

EE2 modulatesp53 but not NER pathways p53, p21, gadd45a [36]

MMR MMR-dependent chromosomal instability from alkylation damage msh6 [53]

Mlh1 deficiency for zebrafish male sterility mlh1 [48]

Completion of meiosis lack ofmlh1 in zebrafish mlh1 [49]

MMRdeficiency does not enhance ENU mutagenesis msh6 [51]

Mutations in MMRdevelop tumors mlh1, mlh2, mlh6 [50]

Cadmium down-regulates msh6 msh6 [68]

Cloning ofmsh2 in zebrafish msh2 [46]

Cloning ofmsh6 in zebrafish msh6 [45]

Zebrafish: chiasmataand interference mlh1 [47]NHEJ SB transpositional recombination by NHEJ pathway dna-pk [69]

Ionizing radiation-induced DNA damage. xrcc5/ku80 [54]

Ku70 protects nervous system from radiation damage xrcc6/ku70 [55]

The role of NHEJ in transgene concatemer formation [57]

ZFN targeted disruption in zebrafish [11]

ZFN minireview [10]

ZFN targeted disruption in zebrafish [14]

ZFNas gene therapy agents [70]

NHEJ involved in LINE retrotransposition zfl2-2, ku70, art, ligIV [56]

Rapidmutation of zebrafishgene by ZFNOPEN [13]

Targeted mutagenesis using customized ZFN [12]

Improved somatic mutagenesis in zebrafish using TALENs [15]

HR Chi-stimulated HR and i ts application in zebrafish [71]

HRand NHEJ in zebrafish [59], [58]

Use of RecA fusion proteins for zebrafish genomic modifications reca [62]

Rad52 and RPA mediated mutagenesis rad52, rpa [61]


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Fig. 1. DNA base excision repairpathway (modified from Fortier et al. [23]). DNA base excision repairpathway. The step numberis 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 flap endonuclease 1 (FEN1) and PCNA (not shown).

factor c (rfc) and pms2 remain to be explored. Expression ofmsh6

relative to msh2 and the tissue distribution ofmsh2 varied with

the stage of development [45] until the relative levels stabilizedat 120 hpf [46]. msh2 tended to be localized in the brain, eyes,

telencephalon, and the fourth ventricle at 12 days post fertilization

(dpf)-48 dpf embryos [46]. On theotherhand, Mlh1 foci detected by

immunofluorescence tended to be in the distal regions of zebrafish

synaptonemal complexes (SCs) [47].

MMR is critical for sperm maturation. Male zebrafish 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 specific genes were per-

formed, eggs fertilized with surviving sperm produced malformed

embryos.

Although fish that are homozygous mutant for the MMRgenesmlh1, msh2, and msh6 developed neoplasms particularly neurofi-

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 zebrafish. Although one might expect that

successful mutagenesis would be enhanced by loss of MMR, defi-

ciency in the MMRgene msh6 did not enhance ENU mutagenesis in

thezebrafish germ line [51]. MMRalso contributed to growth arrest

in response to DNAdamage by alkylating agents [52]. Although the

primary response to alkylation damage occurs viaBER, invivo expo-

sure of zebrafisheggs to alkylating agentsresulted 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 DNAthat 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 zebrafish NHEJ pathway have been cloned [54,55].

Ku80 promoted survival of irradiated cells during embryogenesis.

Radiation-induced apoptosis in the Ku80 knockdown zebrafish 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 zebrafish and mammalian sources. The use ofzebrafish LINEs enabled researchers to demonstrate the importance

of Ku70 and Artemis in chicken B lymphocyte lines defective for

these genes [56]. Furthermore, microinjection of linearized DNA

encoding green fluorescent protein into zebrafish embryos was fol-

lowed directly by fluorescence microscopy after which the DNA

was recovered and sequenced. From these data Dai et al. concluded

that NHEJ is the principal mechanism of exogenous gene integra-

tion in zebrafish [57]. Recently, Liu et al., developed a visual-plus

quantitative analysis systems to studyDNA DSB repairs in zebrafish,

which also showed that NHEJ was predominant among the three

DSB repair pathways (NHEJ,HR andSSA) in zebrafish embryos [58].

NHEJ is the underlying mechanism for generating targeted

mutations by new, highly efficient methods known as ZFNs

(locus specific zing-finger nucleases) or TALENs (transcription


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activator-like effector nucleases) in zebrafish 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, andJak3 [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 finally DNA synthesis is initiated using the intact strand as

the template. HR has been reported in intact zebrafish embryos

at early developmental stages [59] and in zebrafish ES cell cul-

tures [60]. Several attempts to use HR to promote gene targeting in

zebrafish have not met with success [61,62], although one recent

report showed that knockdown of Rad51 decreased HR ability in

zebrafish embryos [58]. Much remains tobe studied in thezebrafish

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 first to be identified, as it is capable of

bypassing thymine dimer CPDs. Unlike the replicative DNA poly-

merases, , and , these polymerases exhibit low fidelity when

copying undamaged DNA [6367]. Some like Rev1, however, insert

a correct base (aC residue) opposite a template G but are inaccu-

rate when synthesizing opposite an AP site or certain other minor

groove adducts. DNA polymeraselambda andDNA polymerase mu,

two additional translesion synthesis polymerases found in other

vertebrates, have not yet been identified in the zebrafish genome.

There is no published work on the translesion polymerases in

zebrafishat this time.Giventhe rapidityof DNAsynthesisin thefirsthours after zebrafish fertilization, it seems likely that the transle-

sion polymerases could play an important role [28].

3. The role of BER pathway in zebrafish embryological

development

The role of the BER pathway in DNA repair (Fig. 1) is reviewed

in the preceding section. In zebrafish unfertilized eggs and early

stage embryos are able to perform at least the first three BER

steps [26]. However, BERin early stage embryos (before 12hpf) has

several unexpected features consistent with rapid cellular prolifer-

ation (15min/cycle for the first 10 division cycles). In early stage

embryos, aphidicolin-sensitive DNA polymerase(s) replace PolB,

which is absent, while the presence of backup Mg2+-dependentendonuclease 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 zebrafish. Zebrafish Apex1

shares the same enzymatic properties as its human orthologue. In

zebrafish,Apex1 protein has 78%homology(64% identity)with that

of the human protein. Full knockdown of Apex1 in early zebrafish

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 efficiently. The advantage of the zebrafish system is that

zebrafish embryos can continue developing for up to seven days

with a non-functional heart. Both western blot and immunohisto-

chemistry using anti-Apex1 antibody show that Apex1 is abundant

in unfertilized eggs and throughout development (Fig. 2) [26].

The use of zebrafish made it possible to discover that Apex1

plays a critical and previously unknown role in cell physiology.

These studies could not have been performedin 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

Creb1proteinand its binding partners,Creb1 binding protein (CBP),

Creb1 modulating protein (Crem)and Targets of Creb1 (Torcs 1 and

3).Similar effects areseen in primary cultures of mouse B cells 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

oxidativedamage to DNAare ongoing processesin every cell. These

events are distinct from double strand breaks unless the breaks are

very closely spaced or the damage is overwhelming. Nevertheless,

oxidative damage requires repair to maintain genome integrity and

stability. DNA damage activates checkpoint mechanisms to arrest

the cell cycle in order to allow time to repair the DNA damage. Ifthe damageis toosevere forefficientrepair, thecell 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

theincidenceof cancer. It is involved in a numberof non-neoplastic

processes including reproduction, metabolism, development, drug

and radiation toxicity and aging [76,79,80].

Zebrafish 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 thestudies involving p53 in zebrafish have involved a link to apopto-

sis engendered b y either UV or IR radiation, both of which generate

DSBs [76]. In that situationp53 mediates apoptosisthrough a linear

pathway involving Bax transactivation, Bax translocation from the

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 zebrafish are Mdm2

[83] and 113p53, an alternative splicing p53 isoform [84].

Instead of acting as a dominant negative for p53 [84],

113p53


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Fig. 2. Analysis of theproteinexpressionlevel of Apex1 at different developmental stages in zebrafish by confocal microscopy. (A and B) Apex1, a maternal protein, is stored

in the unfertilizedegg, as shown by fluorescence microscopy (A,FITC-labeled Apex1) and confocalmicroscopy (B, the merged picture withFITC-labeled Apex1 andpropidium

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 theenlarged view of thesquare region in panel I.) The diploid nuclei areclearly visible with

red color. (K and L) The 3.2kb upstream sequence preceding theATG start codon on the Apex promoter in zebrafishwas amplified and inserted into pEGFP-N3 to construct

the pApexp3k-GFP plasmid. Constructs (2nl, 50ng/l) were micro-injected into 2cell stage embryos and GFPexpressionwas examined at 1dpf by.

differentially modulated p53 target gene expression to antago-

nize p53 apoptotic activity after zebrafish embryos were exposed

to foreign DNA with broken ends or IR that generated DSBs

[81,84]. Overexpression of113p53 enhanced p53-dependent

up-regulation ofp21 and mdm2, but inhibited p53-dependent up-

regulation of the proapoptotic gene bax [76,84].

In zebrafisha common methodology forknocking downselected

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 need notbe processedbut,instead, bindsdirectlyto itscom-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,

nonspecific, offtarget effects occurso thatthe knockdown pheno-

type needs to be confirmed 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 theknockdownin p53mutant (p53M214K/M214K )

fish [88]. In this line, fish 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 and113p53 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 (p53M214K/M214K ) fish and in wild

type fish in which both p53 and Apex1 had been knocked down(Pei&Strauss, unpublished data). Clearly, Apex1 deficiency acti-

vated a p53-dependent pathway but the brain and neural effects

and the loss of Creb1 resulting from Apex1 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.

Inaddition, one mightalso ask whethertheendsof 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

cannotbe degraded readily. Hence,it wouldtrigger a p53-mediated

apoptotic response


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Zebraﬁsh as a Model System to Study DNA Damage and Repair

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