The Drosophila melanogaster DNA Ligase IV Gene Plays a Crucial Role in the Repair of Radiation-Induced DNA Double-Strand Breaks and Acts Synergistically With Rad54

Corresponding author: Albert Pastink, Sylvius Laboratory, LUMC, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands., A.Pastink{at}lumc.nl (E-mail)

Corresponding editor: L. S. SYMINGTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.

DNA double-strand breaks (DSBs) pose a serious threat to the stability of the genome. If left unrepaired, DSBs can cause cell death or contribute to the formation of gross chromosomal rearrangements such as translocations and deletions. A variety of damaging agents such as X rays and chemical compounds such as bleomycine can cause the formation of DSBs. Furthermore, DSBs arise as intermediates during V(D)J rearrangement in differentiating lymphocytes, meiotic recombination, and certain transposition events.

To counteract the deleterious effects of DSBs, two main repair pathways exist in eukaryotes: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR requires the presence of an undamaged homologous DNA that can be used as a template. In this way, HR ensures accurate DSB repair. NHEJ is based on ligation of the two ends and does not require extensive sequence homology. Frequently, NHEJ is associated with insertion or deletion of a few nucleotides at the site of the break (for reviews see PASTINK et al. 2001 ; VAN GENT et al. 2001 ). The relative contribution of both repair pathways depends on the organism, the phase of the cell cycle, the developmental stage, and, presumably, the structure of break formed. In lower eukaryotes, HR is the primary repair mechanism. In the yeast Saccharomyces cerevisiae, the contribution of the NHEJ to the repair of DSBs can be detected only when HR is impaired (MILNE et al. 1996 ; SIEDE et al. 1996 ). In higher eukaryotes, both HR and NHEJ contribute to the repair of DSBs. HR is especially important during the S and G2 phases of the cell cycle when the sister chromatid can be used as a template and during early development (TAKATA et al. 1998 ; ESSERS et al. 2000 ; RICHARDSON and JASIN 2000 ). NHEJ predominates in adult organisms and during the G1 and early S phases of the cell cycle. The NHEJ pathway was first studied in mammals using rodent cell mutants and involves a number of proteins including Ku70, Ku80, DNA-PKcs, Ligase IV and its associated protein XRCC4, and the Artemis protein. The current model of DSB repair by NHEJ assumes that a heterodimer of Ku70 and Ku80 binds to DNA ends and recruits DNA-PKcs to the site of the damage to form an active DNA-PK complex (FEATHERSTONE and JACKSON 1999 ; DOHERTY and JACKSON 2001 ; VAN GENT et al. 2001 ). Binding of Ku to the DNA ends is also required for recruitment of Ligase IV and XRCC4 to the site of the break, which results in stimulation of DNA end ligation (CRITCHLOW et al. 1997 ; GRAWUNDER et al. 1997 ; MCELHINNY et al. 2000 ). The newly identified Artemis protein binds to DNA-PKcs and has endo- and exonucleolitic activities required for processing of DNA ends (MOSHOUS et al. 2001 ; MA et al. 2002 ).

Mice deficient in one of the components of the DNA-PK complex display an increased sensitivity to ionizing radiation and a severe combined immunodeficiency phenotype due to defects in V(D)J recombination (NUSSENZWEIG et al. 1996 , NUSSENZWEIG et al. 1997 ; ZHU et al. 1996 ; GU et al. 1997 ). Mouse embryonic stem cells deficient in DNA-PKcs, however, are not hypersensitive to ionizing radiation (GAO et al. 1998A ; TACCIOLI et al. 1998 ). Evidently, the repair of DSBs by NHEJ is independent of DNA-PKcs in these cells. In yeast and also in Drosophila and Caenorhabditis elegans, no obvious homolog of DNA-PKcs has been identified. The role of DNA-PKcs possibly could be restricted to cells that strongly rely on NHEJ for the repair of X-ray-induced DSBs. In contrast to the loss of DNA-PKcs or the Ku function, inactivation of Ligase IV or XRCC4 in mice results in embryonic lethality as a consequence of massive apoptosis in the central nervous system (BARNES et al. 1998 ; FRANK et al. 1998 ; GAO et al. 1998B ; GRAWUNDER et al. 1998A ). Rescue of embryonic lethality is possible in a p53-, ATM-, or Ku-deficient background (FRANK et al. 2000 ; KARANJAWALA et al. 2002 ). In addition to the repair of DSBs, studies in yeast and mammalian cells indicate that the Ku proteins and DNA-PKcs are also involved in maintenance of telomere length and normal chromosomal DNA end structure (BOULTON and JACKSON 1996A , BOULTON and JACKSON 1998 ; GRAVEL et al. 1998 ; BAILEY et al. 1999 ; HSU et al. 1999 ; CHAI et al. 2002 ).

Drosophila melanogaster has been used extensively to study the mutagenic effects of ionizing radiation (IR) and it represents an attractive system to study DSB repair in a multicellular organism (PASTINK et al. 2001 ). Flies deficient in Rad54 are highly sensitive to X rays and methyl methanesulfonate (MMS), implying that HR contributes significantly to the repair of DSBs in somatic cells (KOOISTRA et al. 1997 ). Inactivation of Rad54 or spindle-B, one of the Rad51 paralogs in Drosophila, leads to defects in meiosis (GHABRIAL et al. 1998 ). Increased MMS sensitivity, as compared with wild-type strains, has not been observed for the spindle-B mutant.

To study the contribution of NHEJ to the repair of DSBs in flies and to investigate the role of DNA Ligase IV in a multicellular organism, we isolated the Drosophila DNA Ligase IV gene, Lig4, and examined its function by generating mutant strains. In contrast to mice, homozygous null flies are viable and show increased sensitivity to ionizing radiation. A strong synergistic effect for radiosensitivity was detected in Lig4; Rad54 double-mutant flies.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila DNA Ligase IV gene analysis:
The Drosophila DNA Ligase IV gene (Lig4) was identified by screening the Berkeley Drosophila Genome Project database (http://www.fruitfly.org) and is located on the X chromosome at position 12A9-B1. A full-length Lig4 cDNA clone (RE37186) was purchased from Research Genetics (Huntsville, AL).

Two-hybrid analysis:
A 773-bp fragment encoding the C-terminal end of Drosophila DNA Ligase IV was amplified by PCR using the Expand High Fidelity PCR system (Roche, Indianapolis) and inserted as a SalI-EcoRI restriction fragment into the single-copy two-hybrid vectors pPC97 and pPC86, carrying the GAL4 DNA-binding domain and the GAL4-activating domain, respectively (CHEVRAY and NATHANS 1992 ). In a similar fashion, a full-length cDNA fragment of 708 bp encoding the Drosophila XRCC4 protein was amplified by PCR and inserted into SalI- and SpeI-digested pPC97 and pPC86. Two-hybrid studies were performed using the S. cerevisiae strain Y190 (HARPER et al. 1993 ). Transfectants were selected for tryptophane and leucine prototrophy on YNB medium [0.76% yeast nitrogen base (Difco, Detroit), 2% glucose] containing 30 mg/liter adenine and 50 mM 3-aminotriazole. Protein-protein interactions were detected using a ß-galactosidase colony filter assay.

Generation of Lig4-deficient flies:
To generate Lig4 mutant flies, the Drosophila EP(X)0385 insertion line CG12176^EP(X)0385 (w¹¹¹⁸ P{w^+mC EP}EP385), abbreviated here as Lig4^EP385, was used. Sequence analysis showed that the site of integration of the EP element is 38 bp upstream of the ATG start codon of the Lig4 gene and is located within the 5'-untranslated region (UTR) of the gene. To mobilize the EP element, we crossed w¹¹¹⁸, Lig4^EP385 females to Sb P [ry⁺ 2-3]/TM3 males. Males from this cross were subsequently crossed to white (w) females. On the basis of the eye color phenotype, four types of females could be distinguished among offspring of the last cross. Only those with an eye color darker than the original bleached eye color of the Lig4^EP385 line (putative insertion mutants) or those with white eyes (putative deletion mutants) were analyzed further. A PCR screen with the EP inverted-repeat primer PTR2 (5'-ACGGGACCACCTTATGTTATTTCATCATG-3') and a Lig4-specific primer LHR2 (5'-GCGATGGCACTGATGTATCC-3'; nucleotides 2955–2975 of the genomic sequence) was used to identify insertion mutants (see Fig 1). The LGF4 forward primer (5'-TGCCGAGGCCTTGCACATCT-3'; nucleotides 364–344 upstream of the ATG start codon) and the LHR2 reverse primer were used to screen the putative deletion mutants. The following PCR conditions were used: 1 min 94°, 1 min 60°, 3 min 72° for 30 cycles.

View larger version (11K): In this window In a new window Download PPT slide   Figure 1. Structure of the Lig4 gene. (A) The intron-exon organization was determined after sequence analysis of a 3029-bp Lig4 cDNA and comparison with the genomic sequence present in the database. The translated region of the Lig4 gene is represented by hatched boxes, and introns and 5'- and 3'-UTRs are represented as open boxes. The ATG start codon is located at position 67. The stop codon, at position 3009, is indicated by an asterisk. To inactivate the Lig4 gene, the Lig4EP385 line carrying an EP element inserted at position 28 was used. After mobilization of the EP element, the LGF4 and LHR2 primers were used to screen for the presence of deletions. (B) Two mutant lines generated and used for phenotypical analysis, Lig45 and Lig457. The deletion in the Lig45 mutant line extends up to nucleotide 805. Lig457 uncovers nearly the entire Lig4 gene extending up to nucleotide 2534, leaving only the last 475 nucleotides of the ORF.

To obtain Lig4-deficient flies, females containing a deletion in Lig4 were crossed to w males. Individual males that could possibly carry the mutation in the Lig4 gene were crossed to Muller 5 females and the female offspring were again screened for the presence of the deletion in the Lig4 gene using the LGF4 and LHR2 primers. Next, w Lig4/Muller 5 females were crossed to Muller 5 males. In the following generation, w Lig4 males were crossed to w Lig4/Muller 5 females to produce flies homozygous-deficient for Lig4. To determine the length of the deletions in the Lig4 gene, PCR products were gel purified, cloned into pGEM-T Easy (Promega, Madison, WI), and sequenced. In total, 18 different Lig4 deletion mutants were generated, of which the Lig4⁵ and Lig4⁵⁷ lines were the subject of phenotypical analysis.

Treatment of Drosophila with DNA-damaging agents:
In Drosophila, the sensitivity to DNA-damaging agents is dependent on the developmental stage and therefore embryos and larvae of different stages were used for treatment. w, Lig4-deficient females were crossed to Lig4-proficient Muller 5 males. After a 4-, 16-, 24-, or 28-hr period of egg laying, embryos and larvae of different developmental stages were treated directly or after further development with increasing doses of X rays. Fly cultures were grown at 25° and after 12–18 days the offspring were scored. In the untreated control, the ratio of Lig4-deficient males to heterozygous females is theoretically 1.0. If the sensitivity to DNA-damaging agents is increased, this ratio will decrease with increasing dose.

To determine the effect of storage in the oocyte of maternal Lig4 protein and/or mRNA (maternal effect), Lig4 heterozygous females were crossed to mutant males and the resulting progeny were tested for hypersensitivity to X rays in comparison to the reciprocal cross.

To investigate the contribution of HR to the repair of X-ray-induced DSBs in early developmental stages, we used the okr⁷⁸² (originally called okr²⁰⁶⁸²) mutant allele of Rad54, named here Rad54⁷⁸². The Rad54⁷⁸² allele carries a single-base-pair change, which results in a threonine-to-isoleucine change at position 660, which is located outside of the helicase domains but within a region conserved among Rad54 homologs (K. MCKIM, personal communication). Unlike null alleles of Rad54, which result in female sterility, homozygous Rad54⁷⁸² females are fertile. JS17/cn Cy males were crossed to Rad54⁷⁸²/Rad54⁷⁸² homozygous mutant females and in a parallel cross Rad54⁷⁸²/Rad54⁷⁸² males were crossed to JS17/cn Cy females. The deficiency chromosome Df(2L)JS17, referred to as JS17, uncovers the Rad54 gene. The offspring were treated with increasing doses of X rays at different stages of development. In this generation, the expected ratio of Rad54⁷⁸²/Rad54⁷⁸² (Cy+) flies and Rad54⁺/Rad54⁷⁸² (Cy) flies is 1:1 according to Mendelian laws. The sensitivity of Rad54⁷⁸²/Rad54⁷⁸² mutant flies was calculated relative to Rad54⁺/Rad54⁷⁸² heterozygous flies.

To determine the relative contribution of NHEJ and HR to the repair of DSBs, Lig4; Rad54 double-mutant flies were generated by crossing Lig4⁵⁷/Lig4⁵⁷; Rad54^A17-11/cn Cy females to JS17/cn Cy males. The Rad54^A17-11 mutation is a null allele of Rad54 due to a GC-to-AT transition at the splice acceptor site of the second intron as has been previously described (KOOISTRA et al. 1997 ). In the untreated offspring, the expected ratio of Lig4⁵⁷; Rad54^A17-11/Rad54^A17-11 males, Lig4⁵⁷; Rad54⁺/Rad54^A17-11 males, Lig4⁺/Lig⁵⁷; Rad54^A17-11/Rad54^A17-11 females, and Lig4⁺/Lig⁵⁷; Rad54⁺/Rad54^A17-11 females is 1:2:1:2. Homozygous cn Cy/cn Cy flies were not recovered due to embryonic lethality. The sensitivity of single and double mutants was calculated relative to Lig4⁺/Lig⁵⁷; Rad54⁺/Rad54^A17-11 heterozygous females. Lig4; Rad54 double mutants and Lig4 and Rad54 single mutants were treated at different developmental stages with increasing doses of X rays or 24–48 hr after egg laying with 0.2 ml/vial of 0.01, 0.02, 0.03, 0.06, or 0.08% MMS in phosphate buffer (pH 6) or with 0.2 ml/vial of 0.01, 0.05, 0.075, or 1.0 mM cis-diamminedichloroplatinum (cisDDP) in water.

X rays were generated with a SMART 225 machine at 200 kV, 4 mA, filter 1 mm Al at dose rates of 1 Gy/min.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sequence analysis of Drosophila DNA Ligase IV gene:
The Drosophila DNA Ligase IV gene, Lig4 (CG12176), was identified by searching the Drosophila Genome Database (http://www.fruitfly.org). The Lig4 gene is located at position 12A9-B1 on the X chromosome. Sequencing of a Lig4 cDNA plasmid clone (RE37186) revealed an insert of 3029 bp. Within this sequence an open reading frame (ORF) from position 67 to 3009 could be recognized. Comparison with the genomic sequence in the database confirmed the presence of the three predicted introns at positions 379–446, 612–670, and 1582–1643 in the Lig4 gene (see Fig 1). Compared with the genomic sequence, base-pair substitutions were observed at positions 2091 (C to A) and 2093 (C to A), resulting in a leucine-to-isoleucine change. The sequence of the start codon AAAATGA matches the initiation consensus in Drosophila (C/A)AA(C/A)ATG very well (CAVENER 1987 ). A putative polyadenylation signal, AATAAA, is present starting at position 3176 in the 3'-UTR of the Lig4 gene.

The predicted 918-amino-acid sequence of Lig4 protein is shown in Fig 2 aligned with human and yeast Lig4 proteins. The most extensive sequence homology is seen in the so-called "core" region conserved between eukaryotic DNA ligases, which includes the five motifs (I–V) that are conserved between ATP-dependent DNA ligases and RNA capping enzymes and the conserved peptide that is found in all the ATP-dependent DNA ligases (WEI et al. 1995 ; reviewed in TOMKINSON and MACKEY 1998 ; MARTIN and MACNEILL 2002 ). Within this region, DmLig4 shares 34% identity (53% similarity) and 25% identity (42% similarity) with hLig4 and ScLig4, respectively. The active site lysine is located at position 282 in the first motif.

View larger version (178K): In this window In a new window Download PPT slide   Figure 2. Sequence alignment of Drosophila DNA Ligase IV (DmLig4), human Ligase IV (hLig4), and Ligase IV from S. cerevisiae (ScLig4). Protein sequences were aligned using the ClustalW algorithm. Identical and similar amino acid residues are indicated by solid backgrounds and shading, respectively, using the Boxshade program (CORPET 1988 ). The active site lysine residue at position 282 is indicated by an arrowhead and the core region conserved between eukaryotic DNA ligases is delineated by a thick line. Within this region the DmLig4 protein displays significant sequence homology with the human and the yeast Lig4 proteins (34 and 25% identity, respectively). The level of identity was determined using the Emboss local pairwise alignment algorithm (http://www.ebi.ac.uk/emboss/align/).

At the C-terminal half of hLig4 and ScLig4, two BRCT (BRCA1 carboxy terminus) domains have been identified. In DmLig4, only one BRCT domain between residues 666 and 752 was recognized using the Prosite database (http://www.expasy.org/prosite/). The second BRCT domain, typically located at the C terminus, could not be identified. Although BRCT domains have been implicated in protein-protein interactions, human Lig4 binds to XRCC4 via a motif located between rather than within the BRCT domains (GRAWUNDER et al. 1998B ). On the other hand, data obtained by HERRMANN et al. 1998 suggest that in the case of ScLig4 the second BRCT domain is required for binding of ScLig4 to Lif1 (yeast XRCC4). To study the interaction between Lig4 and the putative XRCC4 protein from Drosophila identified on the basis of homology by SIBANDA et al. 2001 , two-hybrid studies were carried out. A strong interaction was observed between the C-terminal part of the Lig4 protein fused to the GAL4 DNA-binding domain (bait vector) and the XRCC4 fused to the activation domain (prey vector) of the GAL4 transcription factor. The presence of a conserved BRCT domain at the C-terminal end of Lig4 is apparently not required for binding to XRCC4. This strong interaction clearly demonstrates the presence of a functional XRCC4 homolog in Drosophila. In the reciprocal two-hybrid experiment when the Lig4 fragment was cloned in the prey vector and the XRCC4 cDNA in the bait vector, only a very weak interaction was seen. Unidirectional interactions have been observed previously using the yeast two-hybrid assay. For example, complex formation between Rad51 and Rad54 proteins can be detected in only one direction (CLEVER et al. 1997 ; GOLUB et al. 1997 ).

Generation of Lig4-deficient flies:
To study the role of Lig4 in the repair of DSBs, mutant flies were generated by P-element mutagenesis. The EP line Lig4^EP385 contains a single P insertion in the 5'-UTR of the Lig4 gene. To mobilize the EP element, Lig4^EP385 females were crossed to Sb P [ry⁺ 2-3]/TM3 males. The male offspring were crossed to white females, and among the female offspring we selected newly induced insertion and deletion mutants on the basis of eye color phenotype (see MATERIALS AND METHODS). Among 800 females with an eye color darker than that of the original EP line, no EP insertions in the Lig4 gene were found. Screening of 200 white-eyed females and subsequent analysis of mutations resulted in the identification of 18 different Lig4 deletion mutants. All the mutants were analyzed by sequencing. Since the LGF4 primer is located only 364 bp upstream of the original EP insertion site, the left side of each deletion maps relatively close to the original integration site. The right side of each deletion maps within the ORF of the Lig4 gene. Two of these Lig4 deletion mutants were used for the phenotypical analysis. The deletion in the Lig4⁵ mutant line extends until nucleotide 805 of the Lig4 gene, completely deleting the first two exons. The Lig4⁵⁷ mutant carries the largest deletion generated in our screen. It uncovers nearly the entire Lig4 gene until nucleotide 2534, deleting three of four exons and leaving only 475 nucleotides of the ORF (Fig 1B).

Lig4-deficient mutants are viable and fertile as wild-type flies and do not show any signs of abnormal phenotype. Lig4-deficient males emerge in a nearly 1:1 ratio with their heterozygous sisters, indicating no measurable developmental retardations.

Lig4 mutant flies are sensitive to ionizing radiation:
Homozygous Lig4-deficient females were crossed to (Lig4-proficient) Muller 5 males. In the F₁, the expected ratio of the Lig4-deficient males to heterozygous females is 1.0. If the Lig4 deficiency results in increased sensitivity to DNA-damaging agents, this ratio will decrease with increasing dose given to the F₁ embryos and larvae. To determine the X-ray sensitivity of the original Lig4^EP385 line as well as of the Lig4⁵⁷ and Lig4⁵ mutant lines, 0- to 24-hr embryos, 24- to 48-hr larvae, and 48- to 72-hr-old larvae were exposed to a dose of 9 Gy (Fig 3A). The Lig4^EP385/Lig4^EP385 line itself displayed a limited hypersensitivity to X rays at the embryonic stage. The ratio of males to females was 0.85 (53/62) compared to 1.08 (329/305) for the untreated control. The larval stages of the Lig4^EP385/Lig4^EP385 line showed hardly any sensitivity to X rays in comparison to the heterozygous Lig4⁺/Lig4^EP385. The ratios of males to females for 24- to 48-hr- and 48- to 72-hr-old larvae were 0.91 (300/330) and 1.02 (399/391), respectively. Apparently, the insertion of the EP element in the 5'-UTR of the Lig4 gene does not severely interfere with the expression of the gene.

View larger version (47K): In this window In a new window Download PPT slide   Figure 3. Effect of X rays on Lig4-deficient mutants. On the y-axis the ratio of Lig4-deficient males to Lig4-proficient females is indicated. According to Mendelian laws, the expected ratio is 1.0. If the Lig4 defect results in increased sensitivity to X rays, this ratio will drop. (A) Lig4EP385 (EP385) (solid bar), Lig457 (striped bar), and Lig45 (open bar) strains were exposed to a dose of 9 Gy at different stages of development (hours after egg laying). (B) Contribution of Lig4 to the repair of X-ray-induced DSBs at embryonic and larval stages of development for Lig457 (solid bar) and Lig45 (open bar) after a dose of 9 Gy. (C) Dose-dependent decrease in recovery of Lig457 and Lig45 mutant lines (combined data). Nonirradiated controls are indicated as C or 0 Gy. Standard deviations are based on the total number of flies scored.

The two mutant lines generated in our screen, Lig4⁵ and Lig4⁵⁷, were hypersensitive to X rays (Fig 3A). For the nonirradiated controls, the ratios of males to females were 0.98 (538/550) and 0.89 (220/246) for the Lig4⁵⁷ and Lig4⁵ mutant lines, respectively. Lig4⁵⁷ and Lig4⁵ embryos that were 0–24 hr old showed a 4.1-fold [0.24 (82/343)] and a 4.7-fold [0.19 (14/74)] increase in sensitivity in comparison to the nonirradiated control, respectively. At later stages of development, the hypersensitivity of larvae to X rays gradually decreases. Larvae that were 24–48 and 48–72 hr old showed ratios of 0.68 (411/603) and 1.0 (665/660) for the Lig4⁵⁷ mutant and 0.43 (119/279) and 0.76 (226/296) for the Lig4⁵ mutant. Both the Lig4⁵⁷ and Lig4⁵ mutant lines were equally hypersensitive to X rays at the embryonic stage of development, indicating that both are null mutants.

To investigate the contribution of NHEJ to the repair of DSBs at early stages of development, 0- to 4-hr- and 4- to 20-hr-old embryos and larvae and 28- to 44-hr-old larvae were exposed to a dose of 9 Gy (Fig 3B). Surprisingly, early embryos (0–4 hr) did not display an enhanced sensitivity to X rays. The ratios of males to females obtained for the Lig4⁵⁷ and Lig4⁵ strains were 0.98 and 0.93, respectively. These values hardly deviated from those obtained from the untreated controls (1.01 and 0.9, respectively). However, in 4- to 20-hr-old embryos a strong increase in sensitivity to X rays was seen. In Lig4⁵⁷ and Lig4⁵ mutant lines, a ratio of 0.14 (77/570) and 0.15 (77/500) was obtained for 4- to 20-hr-old embryos, resulting in a 7.2-fold (1.01/0.14) and a 6-fold (0.90/0.15) increase in sensitivity, respectively. In the case of 28- to 44-hr-old larvae, the hypersensitivity to X rays was less pronounced.

The sensitivity of the Lig4⁵⁷ and Lig4⁵ mutant lines (data combined) to different doses of X rays was assessed at different stages of development (Fig 3C). At the most sensitive stage (4- to 20-hr embryos), the ratio of Lig4-deficient males to Lig4-proficient females was 0.56 after 6 Gy and 0.15 after a dose of 9 Gy. At the age of 28–44 hr, hypersensitivity was seen only after exposure to the highest dose. Again, 0- to 4-hr embryos did not exhibit an increased sensitivity to X rays.

Maternal effects contribute during the first 24 hr of development:
In early Drosophila embryos, the repair of DNA damage is also dependent on maternal factors deposited in the egg. To determine the effect of storage in the oocyte of maternal Lig4 protein and/or mRNA, Lig4 heterozygous females were crossed to mutant males. In the offspring, the ratio of mutant males and heterozygous females was determined and compared to the ratio of mutant males to heterozygous females obtained after crossing Lig4⁵⁷-deficient females to Lig4-proficient males (Fig 4). After treatment of 0- to 8-hr-old embryos and 8- to 24-hr embryos and larvae, the recovery of males originating from Lig4-deficient females was decreased in comparison with males obtained from Lig4-proficient females. Treatment of 0- to 8-hr-old embryos with a dose of 9 Gy resulted in a 2-fold difference in sensitivity (ratios 0.76/0.39) of males originating from Lig4⁺/Lig4⁵⁷ females compared to those coming from Lig4⁵⁷/Lig4⁵⁷ females (Fig 4A). Treatment of 8- to 24-hr-old embryos and larvae with the same dose of X rays resulted in a 3.3-fold difference in sensitivity (0.69/0.21; Fig 4B). After 24–48 hr of larval development, no difference in sensitivity was seen among the offspring obtained from the two crosses (Fig 4C). Apparently, at this stage the maternally deposited Lig4 protein and/or mRNA is exhausted. At a dose of 9 Gy, the ratio dropped to 0.74 for both crosses, and at a dose of 15 Gy, dropped even further to 0.23 and 0.16 for males derived from Lig4^-/Lig4^- and Lig4⁺/Lig4^- females, respectively.

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. Maternal contribution of Lig4 activity. Maternal effects were measured as the difference in recovery of Lig4-deficient males coming from Lig4-deficient females crossed to M5 males () to those coming from heterozygous Lig4 females crossed to Lig4-deficient males (•). Flies were irradiated with increasing doses of X rays (A) 0–8 hr, (B) 8–24 hr, and (C) 24–48 hr after egg laying. Standard deviations are based on the total number of flies scored.

HR repairs DSBs during very early embryonic stages of fly development:
The data depicted in Fig 3B and Fig C, indicate that NHEJ hardly contributes to the repair of X-ray-induced DNA damage in very early embryos. To investigate the contribution of HR to the repair of X-ray-induced DSBs at these stages, we used the Rad54⁷⁸² allele of Rad54 (see MATERIALS AND METHODS). Rad54⁷⁸²/Rad54⁷⁸² females were crossed to JS17/cn Cy males and in a parallel control cross JS17/cn Cy females were mated to Rad54⁷⁸²/Rad54⁷⁸² males. The sensitivity of the JS17/Rad54⁷⁸² (Rad54^-/-) mutant was calculated relative to Rad54⁷⁸²/cn Cy (Rad54^+/-) heterozygous flies. Sensitivity of different developmental stages to X rays (0–8, 8–24, and 24–48 hr) was tested with increasing doses. In nonirradiated controls, the observed ratios were almost equal to the expected 1:1 ratio (Fig 5). In the first 8 hr of embryonic development, the ratio of Cy⁺ (Rad54^-/-) to Cy (Rad54^+/-) flies in the offspring of Rad54⁷⁸²/Rad54⁷⁸² females decreased with increasing dose. In contrast, the ratio of Cy⁺ to Cy flies in the offspring of heterozygous JS17/cn Cy females did not decrease with dose. At a dose of 9 Gy, nearly a fourfold difference in sensitivity was observed between the Rad54-deficient offspring from Rad54⁷⁸²/Rad54⁷⁸² females in comparison to the offspring from JS17/cn Cy females (Fig 5A). Among 8- to 24-hr-old embryos and larvae, no difference in sensitivity was observed between Rad54-deficient offspring from Rad54⁷⁸²/Rad54⁷⁸² females or JS17/cn Cy females (Fig 5B). Similar results were obtained for 24- to 48-hr-old larvae exposed to increasing doses of X rays (Fig 5C). The results suggest that after 8 hr of development the contribution of maternal Rad54 protein and/or mRNA is strongly reduced or exhausted. The significant increase in X-ray sensitivity seen in 24- to 48-hr larvae indicates also that at later stages of development HR plays an important role in the repair of X-ray-induced DSBs.

View larger version (23K): In this window In a new window Download PPT slide   Figure 5. Effect of X rays on Rad54-deficient mutants. The role of Rad54 in the repair of X-ray-induced DSBs was determined using the Rad54782 allele of Rad54. The sensitivity to X rays is given as the ratio of Rad54-deficient to Rad54-proficient flies. Maternal effects are seen as the difference in recovery of Rad54-deficient flies coming from Rad54782/Rad54782 females crossed to JS17/cn Cy males () in comparison to those coming from JS17/cn Cy females crossed to Rad54782/Rad54782 males (•). Flies were exposed to X rays (A) 0–8 hr, (B) 8–24 hr, and (C) 24–48 hr after egg laying. Standard deviations are based on the total number of flies scored.

NHEJ and HR act synergistically in the repair of X-ray-induced DSBs:
To determine the relative contribution of NHEJ and HR to the repair of DSBs, Lig4; Rad54 double-mutant flies were generated by crossing Lig4⁵⁷/Lig4⁵⁷; Rad54^A17-11/cn Cy females to Lig4⁺; JS17/cn Cy males. In this experiment only the Lig4⁵⁷ strain was used, since the initial survival experiments did not show a difference between the Lig4⁵⁷ and Lig4⁵ strains. The sensitivity of single and double mutants was calculated relative to Lig4^+/-; Rad54^+/- heterozygous females (see MATERIALS AND METHODS). The embryos and larvae were treated with increasing doses of X rays at different developmental stages. After exposure of 0- to 24-hr-old embryos and larvae to a dose of 3 Gy, a 3.2-fold and a 2.5-fold increase in hypersensitivity of the Lig4; Rad54 double mutant was observed in comparison to the Rad54 and Lig4 single mutants, respectively (Fig 6A). At a dose of 6 Gy, Lig4; Rad54 double-mutant flies displayed a 10-fold and a 4-fold increase in sensitivity in comparison to Rad54 and Lig4 single mutants, respectively. In 0- to 24-hr-old embryos and larvae, the difference in sensitivity observed between Lig4 and Rad54 single mutants can be partially ascribed to the maternal effect in the case of Rad54. Twenty-four hours later (24–48 hr), exposure to a dose of 3 Gy resulted in 12.5-fold and 11-fold increases in sensitivity of the Lig4; Rad54 double mutant in comparison to Rad54 and Lig4 single mutants, respectively (Fig 6B). At higher doses, the toxic effect of the X rays becomes more severe and double-mutant as well as Rad54 single-mutant flies were not recovered anymore. Treatment of 48- to 72-hr-old larvae with a dose of 3 Gy resulted in a 4-fold increase in sensitivity of the double mutant in comparison to both single mutants (Fig 6C). At a dose of 6 Gy and higher, both the Lig4; Rad54 and Rad54 larvae were killed. Only a relatively small increase in sensitivity is seen in Lig4 larvae exposed to higher doses of X rays (Fig 6C). At 72–96 hr after egg laying, irradiation with a dose of 3 Gy did not result in an increased sensitivity of the single and double mutants. At higher doses, the Rad54 single mutant and the Lig4; Rad54 double mutant both showed the same drastic increase in sensitivity. The effects of increasing doses of X rays on the Lig4 single mutant were much less severe (Fig 6D).

View larger version (29K): In this window In a new window Download PPT slide   Figure 6. Sensitivity of Lig4; Rad54 double-mutant flies to X rays. Double-mutant flies were generated by crossing Lig457/Lig457; Rad54A17-11/cn Cy females to JS17/cn Cy males. In the untreated offspring, the ratio of Lig4; Rad54 double-mutant males, Lig4 single-mutant males, Rad54 single-mutant females, and Lig4; Rad54 heterozygous females is expected to be 1:2:1:2 according to Mendelian laws. The ratio of single and double mutants was calculated relative to Lig4; Rad54 heterozygous females. The ratios for the Lig4 mutants are indicated on the left y-axis and the ratios for the Rad54 mutants and the Lig4; Rad54 double mutants are indicated on the right y-axis. The offspring were exposed to increasing doses of X rays (A) 0–24 hr, (B) 24–48 hr, (C) 48–72 hr, and (D) 72–96 hr after egg laying. (•) Lig4- Rad54+/-, () Lig4+/- RAD54-/-, and () Lig4- Rad54-/-. Standard errors are based on fly count per vial.

The results shown in Fig 6C and Fig D, indicate that at later stages of larval development the hypersensitivity of Lig4 mutants is less pronounced. After a dose of 9 Gy, no Lig4; Rad54 double-mutant flies could be recovered anymore. Exposure of Lig4 mutant larvae of 48–72 and 72–96 hr resulted in a moderate increase in sensitivity at the higher doses applied. After a dose of 30 Gy, the ratio dropped to 0.24 and 0.37 for 48- to 72-hr and 72- to 96-hr larvae, respectively. These data indicate that after 48 hr of development, the role of NHEJ in the repair of X-ray-induced DSBs becomes less important than the role of HR.

Lig4-deficient flies are not sensitive to MMS and cisDDP:
As previously described, the Rad54 mutant flies display a strong hypersensitivity to the alkylating agent MMS and to the crosslinking agent cisDDP (KOOISTRA et al. 1999 ). To determine if NHEJ is also involved in the repair of DSBs resulting indirectly as a consequence of exposure to these agents, Lig4⁵⁷/Lig4⁵⁷; Rad54^A17-11/cn Cy females were crossed to JS17/cn Cy males and the offspring were treated with MMS or cisDDP (see MATERIALS AND METHODS). Exposure of larvae at the age of 24–48 hr to increasing doses of cisDDP or MMS resulted in a nearly equal increase in sensitivity of the Lig4; Rad54 double mutant and the Rad54 single mutant (see Fig 7A and Fig B). Treatment of the Lig4 single mutant with the same doses of cisDDP or MMS resulted in only an increased sensitivity at the highest doses used, suggesting no or a very minor role of NHEJ in the repair of crosslinks or alkyl lesions.

View larger version (16K): In this window In a new window Download PPT slide   Figure 7. Sensitivity of Lig4; Rad54 double-mutant flies to cisDDP and MMS. Mutant flies were generated by crossing Lig457/Lig457; Rad54A17-11/cn Cy females to Lig4+; JS17/cn Cy males. The ratio of single and double mutants was calculated relative to Lig4; Rad54 heterozygous females. The ratios for the Lig4 mutants are indicated on the left y-axis and the ratios for the Rad54 mutants and Lig4; Rad54 double mutants are indicated on the right y-axis. After 24–48 hr of egg laying, larvae were exposed to increasing doses of cisDDP (A) or MMS (B). Standard errors are based on fly count per vial.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In higher eukyarotes, DNA Ligase IV is an essential protein used for the repair of DSBs via NHEJ. Inactivation of the DNA Ligase IV gene in mice results in embryonic lethality due to massive apoptosis in the central nervous system (BARNES et al. 1998 ; FRANK et al. 1998 ; GRAWUNDER et al. 1998A ), which could be rescued in a p53- or ATM-deficient background or by deleting another member of the same repair pathway, namely Ku80 (FRANK et al. 2000 ; KARANJAWALA et al. 2002 ). In humans, partial defects in DNA Ligase IV result in developmental delay and immunodeficiency. Cells derived from patients suffering from this so-called LIG4 syndrome display increased sensitivity to ionizing radiation and impaired repair of DSBs (JEGGO and CONCANNON 2001 ). To examine the effects of null mutations in the DNA Ligase IV gene at the organismal level, we employed D. melanogaster as a system. Sequence analysis of the Drosophila DNA Ligase IV gene, Lig4, revealed an ORF encoding a putative 918-amino-acid protein, which displays extensive sequence homology to other eukaryotic DNA Ligase IV proteins (TOMKINSON and MACKEY 1998 ). Within the so-called core region, DmLig4 shares 34 and 25% identity with hLig4 and ScLig4, respectively.

Lig4-deficient flies were generated by P-element-mediated mutagenesis (see MATERIALS AND METHODS). Two of the deletion mutant lines isolated, Lig4⁵ and Lig4⁵⁷, were characterized in more detail. The Lig4⁵ deletion extends until nucleotide 805 of the genomic sequence and the Lig4⁵⁷ deletion until nucleotide 2534 (see Fig 1). In contrast to LIG4 mutant mice, flies deficient for Lig4 are viable. Both males and females are fertile and show no obvious signs of defects or other abnormalities.

To investigate the role of Lig4 in DNA repair, Lig4-proficient males were crossed to homozygous mutant females and the offspring exposed to DNA-damaging agents. The two mutant lines, Lig4⁵⁷ and Lig4⁵, were equally hypersensitive to IR. The hypersensitivity was most severe after 4 hr of embryonic development. Treatment of 4- to 20-hr-old embryos and larvae with a dose of 9 Gy resulted on average in a sevenfold increase in sensitivity. At later stages of development, the hypersensitivity of Lig4-deficient flies to IR is less severe.

Exposure of very young (0–4 hr) Lig4-deficient embryos to IR did not result in an increase in sensitivity (Fig 3B). Together these results imply that NHEJ contributes significantly to the repair of DSBs inflicted by IR but not during the first hours after fertilization. One possibility is that during early development DSBs are repaired through HR. By using the fertile Rad54⁷⁸² allele, we showed that HR is effective in the repair of DSBs in the first few hours of embryonic development (Fig 5).

To investigate the relative contribution of NHEJ and HR to the repair of DSBs in more detail, Lig4; Rad54 double-mutant flies were generated by crossing Lig4⁵⁷/Lig4⁵⁷; Rad54^A17-11/cn Cy females to JS17/cn Cy males. Surprisingly, the Lig4; Rad54 double-mutant flies were viable. When treated at early developmental stages (0–24 hr), the double-mutant flies were far more sensitive to IR than were either of the single mutants. These results indicate that in 0- to 24-hr-old embryos HR and NHEJ both contribute to the repair of IR-induced DSBs. Twenty-four hours later, when the maternal effect of Rad54 wears off, a strong synergistic effect was observed in the double mutant. At higher doses, the toxic effect of IR becomes very severe and the double-mutant flies do not survive at all. Larvae that are 48–72 and 72–96 hr old rely predominantly on the HR for the repair of IR-induced DSBs, as shown by the relatively small increase in sensitivity seen for the Lig4-deficient flies. When treated with MMS or cisDDP, hardly any effect of Lig4 deficiency was seen. Only at relatively high doses was a moderate increase in sensitivity observed (see Fig 7). Also, in yeast and humans NHEJ is not required for the repair of alkyl damage and crosslinks in DNA.

The survival data of double-mutant flies demonstrate that in Drosophila both NHEJ and HR contribute significantly to the repair of DSBs induced by ionizing radiation. The data also indicate that with the exception of 0- to 4-hr embryos, both mechanisms can partially compensate for each other. At later stages of development (48–96 hr) the analysis of the double mutant suggests a less important role for NHEJ (see Fig 6C and Fig D). NHEJ and HR have been presented as competing pathways. Binding of Ku or Rad52 proteins to DNA ends at the site of the break would initiate DSB repair through NHEJ or HR, respectively (VAN DYCK et al. 1999 ). The result of such a competition is influenced by the relative amount of Ku70 and Rad52 (or by a functionally related protein in Drosophila, since a structural Rad52 homolog has not been identified), structure of the DSB, cell cycle phase, and stage of development (HIOM 1999 ). The pathway that is used has important consequences for the integrity of the genetic information of an organism. NHEJ is frequently associated with loss or gain of a few nucleotides. Correct restoration of the original sequence can occur via HR if the sister chromatid is used as a template. Using the homologous chromosome as a template could lead to loss of heterozygosity. Early embryonic development in Drosophila is a very rapid process. After fertilization the zygote nucleus undergoes nine divisions in a common cytoplasm to produce a multinucleate syncytium. After migration to the periphery of the egg, the nuclei undergo four more divisions before a cellular membrane is formed and somatic cells are produced. This process takes only 2.5 hr. To avoid accumulation of mutations during the rapid early divisions, which may have deleterious consequences at adult stages, it is beneficial to use HR as the principal mechanism in early development. Studies in mice also indicate that HR is especially important in early development (ESSERS et al. 2000 ). In contrast to mouse embryonic stem cells, a contribution of NHEJ cannot be detected in 0- to 4-hr-old Drosophila embryos, although we cannot exclude the possibility that defects in NHEJ can be fully compensated by HR in contrast to later stages. After the first 4 hr of embryonic development NHEJ does play an important role in the repair of DSBs. Between 4 and 20 hr of development Lig4-deficient flies are most sensitive to increasing doses of ionizing radiation (Fig 3B and Fig C). The hypersensitivity of Lig4-deficient larvae to IR is gradually reduced at later stages of development, indicating that the majority of the radiation-induced DSBs are repaired by HR and only a small fraction by NHEJ. It is difficult to speculate whether it is a competition between the repair pathways that causes those shifts or whether yet another repair system is active at later stages. Later in development the cell divisions definitely become much slower so it is not a matter of cell cycle stage and/or template availability, which would preferentially shift the repair toward HR. These observations differ from the data obtained from mouse studies. Mice deficient for RAD54 are hypersensitive only at very early embryonic stages. In adult mice no hypersensitivity to ionizing radiation was seen in contrast to mice deficient in NHEJ (ESSERS et al. 1997 , ESSERS et al. 2000 ).

The viability of the Lig4; Rad54 mutant flies, as well as survival after low levels of X-ray irradiation, could be explained by evasion of checkpoint control and/or escape from checkpoint-triggered apoptosis at certain stages of the cell cycle or of development. Another possibility is that undamaged dividing cells in the imaginal discs can compensate for the loss of damaged and/or apoptotic cells. The viability of the double mutant after irradiation could also suggest the presence of another repair pathway that partially compensates for the impaired HR and NHEJ mechanisms. One possibility is single-strand annealing (SSA). This mechanism relies on the annealing of repeated sequences on both sides of the DNA break after the formation of 3'-single-strand tails (for review see PASTINK et al. 2001 ). Evidence for the existence of SSA in Drosophila has been recently shown (RONG and GOLIC 2000 ; PRESTON et al. 2002 ). Another mechanism that possibly can overcome IR-induced DSBs in double-mutant flies is microhomology-dependent end joining (µEJ). Evidence for the existence of this pathway has been obtained from studies of mammalian cells mutated in one of the components required for NHEJ (FEENEY 1992 ; GERSTEIN and LIEBER 1993 ; GOTTLICH et al. 1998 ; KABOTYANSKI et al. 1998 ; VERKAIK et al. 2002 ). Studies in yeast using engineered substrates transfected into ku70/rad52 or ku80/rad52 double mutants indicate the existence of a repair mechanism, which repairs DSBs on the basis of microhomology present on both sides of the break (BOULTON and JACKSON 1996A , BOULTON and JACKSON 1996B ). Since small repeated sequences are frequently associated with the formation of chromosomal aberrations in mammalian cells, it will be of great interest to investigate the µEJ pathway in more detail in eukaryotic organisms, including Drosophila. The availability of Lig4 and Rad54 single and double mutants allows us to pursue further studies into the mechanisms of HR and NHEJ.

	FOOTNOTES

¹ Deceased.
² Present address: Department of Molecular and Cellular Biology, Leiden University Medical Center (LUMC), 2333 AL, Leiden, The Netherlands.

	ACKNOWLEDGMENTS

We thank Kim S. McKim for a gift of okr⁷⁸² mutant flies and M. Z. Zdzienicka for critical reading of the manuscript. We are grateful to M. Nivard and other members of the Drosophila group for stimulating discussions and moral support.

Manuscript received June 26, 2003; Accepted for publication August 21, 2003.

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