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. 2013;8(3):e59866.
doi: 10.1371/journal.pone.0059866. Epub 2013 Mar 28.

The Smc5/Smc6/MAGE complex confers resistance to caffeine and genotoxic stress in Drosophila melanogaster

Xiao Li  1 , Ran Zhuo, Stanley Tiong, Francesca Di Cara, Kirst King-Jones, Sarah C Hughes, Shelagh D Campbell, Rachel Wevrick
Affiliations

The Smc5/Smc6/MAGE complex confers resistance to caffeine and genotoxic stress in Drosophila melanogaster

Xiao Li et al. PLoS One. 2013.

Abstract

The SMC5/6 protein complex consists of the Smc5, Smc6 and Non-Smc-Element (Nse) proteins and is important for genome stability in many species. To identify novel components in the DNA repair pathway, we carried out a genetic screen to identify mutations that confer reduced resistance to the genotoxic effects of caffeine, which inhibits the ATM and ATR DNA damage response proteins. This approach identified inactivating mutations in CG5524 and MAGE, homologs of genes encoding Smc6 and Nse3 in yeasts. The fact that Smc5 mutants are also caffeine-sensitive and that Mage physically interacts with Drosophila homologs of Nse proteins suggests that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6 and MAGE are hypersensitive to genotoxic agents such as ionizing radiation, camptothecin, hydroxyurea and MMS, consistent with the Smc5/6 complex serving a conserved role in genome stability. We also show that mutant flies are not compromised for pre-mitotic cell cycle checkpoint responses. Rather, caffeine-induced apoptosis in these mutants is exacerbated by inhibition of ATM or ATR checkpoint kinases but suppressed by Rad51 depletion, suggesting a functional interaction involving homologous DNA repair pathways that deserves further scrutiny. Our insights into the SMC5/6 complex provide new challenges for understanding the role of this enigmatic chromatin factor in multi-cellular organisms.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Eye phenotypes in caffeine-sensitive mutant flies.
(A) Caffeine-dependent eye phenotype of Smc6 (jnj) and MAGE (sst) mutants. Fly genotypes are as follows. Control: EGUF/+; FRT82B +/FRT82B GMR-hid. Smc6 (loss of Smc6 in eye cells): EGUF/+; FRT82B jnjR1/FRT82B GMR-hid. MAGE (loss of MAGE in eye cells): EGUF/+; FRT82B sstRZ/FRT82B GMR-hid. (B-D) Smc6, MAGE or Smc5 homozygous, trans-heterozygous or hemizygous mutants have reduced survival when raised in media with caffeine. Bars represent the survival index (p) and error bars represent SEM. “□” indicates flies eclosed from the same cross. Absence of a bar indicates no surviving flies. Wildtype control flies are w1118. (B) Smc6 mutants are sensitive to caffeine. R1 (jnjR1) is an allele from the caffeine screen, X1 (jnjX1) was generated by an imprecise excision of a P-element adjacent to the 5′UTR of Smc6, and Df (Df(3R)Exel6198) is a deficiency chromosome uncovering the Smc6 locus. (C) MAGE mutants are sensitive to caffeine. RZ (sstRZ) is an allele from the caffeine screen, XL (sstXL) is a targeted knockout, and Df (Df(3R)Antp1) is a deficiency chromosome uncovering the MAGE locus. (D) Smc5 mutants are sensitive to caffeine. Both P5 (Smc5P{GSV1}GS3245) and P7 (Smc5P{GSV6}GS14577) contain P-element insertions in a coding exon of Smc5, and Df (Df(3L)BSC418) is a deficiency chromosome uncovering the Smc5 locus.
Figure 2
Figure 2. Overview of Smc6, MAGE, and Smc5 gene location, structural organization and mutant alleles.
(A) Smc6 is a 14 exon gene located on 3R:95E8–95F1. jnjR1 contains a 4 bp deletion in the 2nd coding exon. jnjX1 contains a 473 bp deletion of sequences upstream of exon 1 (196 bp), the entire exon 1 (252 bp), and a portion of intron 1 (25 bp), with a 12 bp vestige of the original P element remaining. Smc6 genomic locus (3R:20,014,770.20,019,145 [−]) is shown. (B) MAGE is a single exon gene located on the right arm of the 3rd chromosome at position 84C7–84C7. sstRZ has a point mutation that converts a glutamine at position 109 to a stop codon. sstXL carries a targeted deletion of the entire coding sequence of MAGE. MAGE genomic locus (3R:2,979,960.2,980,898 [−]) is shown. (C) Smc5 is a 16 exon gene located in 78D6–78D7 of the left arm of the 3rd chromosome. Exons encoding the longest transcripts are shown. Both P{GSV1}GS3245 and P{GSV6}GS14577 are inserted in the second coding exon. The Smc5 genomic locus (3L:21,562,309.21,566,623 [+]) is shown. CDS, coding sequence.
Figure 3
Figure 3. Mage is part of the Drosophila Smc5/6 complex.
(A) Diagram of a generic Smc5/6 complex in S. pombe (adapted from [70]). The structure in S. cerevisiae is different in that Nse5/6 were found to bind at the hinge. (B) Mage interacts with Nse4 when both proteins are co-expressed in S2 cells. HA-Nse4 co-immunoprecipitated (co-IP) with FLAG-Mage from an S2 cell lysate when two proteins were co-expressed; FLAG-Mage co-IPed with HA-Nse4 from the S2 cell lysate when two proteins were co-expressed. (C) Recombinant Mage interacts with Nse4 and Nse1 directly. Immobilized maltose binding protein (MBP)-fused MAGE or MBP were incubated with 35S-methionine labeled Mage, Nse4, Nse1, or luciferase (as a negative control), respectively. Proteins that were associated with immobilized MBP-Mage or MBP were resolved with SDS-PAGE and visualized by autoradiography. Results show that Mage, Nse4, and Nse1 each interact with MBP-Mage but not with MBP and luciferase does not interact with either of these proteins. (D) Coomassie staining of protein immobilized on 10 µl of amylose beads showed that approximately equal amounts of MBP-Mage and MBP proteins were immobilized on resin beads.
Figure 4
Figure 4. Caffeine exposure results in apoptosis in eye discs of MAGE and Smc6 mutants.
(A) Anti-cleaved-caspase-3 antibody staining of eye discs from third instar larvae of control (WT, FRT82B), MAGE (sstRZ/sstXL), and Smc6 (jnjX1/jnjR1) genotypes raised in either standard media (0 mM caffeine) or media supplemented with 2 mM caffeine for 12 hours before dissection. Images are single stacks of confocal images. More cleaved-caspase-3 foci in eye discs of sstRZ/sstXL and jnjX1/jnjR1 larvae were observed after caffeine exposure. A narrow band of apoptotic cells (white arrow heads) anterior to the presumptive morphogenetic furrow are most noticeable. Scale bar represents 50 µM. (B-D) Quantification and comparison of cleaved caspase-3 staining levels in WT (B), MAGE (C) or Smc6 (D) eye discs, comparing the no caffeine and 2 mM caffeine groups. Data represent mean area stained from multiple eye discs for each genotype per treatment. A maximum projection of all stacks of a confocal image was used to quantify the signal intensity of staining. This value was divided by the area of each eye disc to obtain a ratio representing the relative amount of immunostaining. Error bars represent SEM. A non-paired two-tailed t-test was used to determine statistical significance. **, P = 0.006, ***, P<0.0001.
Figure 5
Figure 5. Smc5/6 mutants are hypersensitive to ionizing radiation.
(A–C) Smc6, MAGE or Smc5 homozygous, trans-heterozygous or hemizygous mutants have reduced survival when exposed to 40 Gy of IR. Bars represent the survival index (p) ± SEM. “□” indicates flies eclosed from the same cross. Absence of a bar indicates that no flies survived at that IR dose. (A) Smc6 mutants are hypersensitive to IR. R1 (jnjR1) and X1 (jnjX1) are Smc6 alleles. Df (Df(3R)Exel6198) is a deficiency chromosome uncovering the Smc6 locus. (B) MAGE mutants are hypersensitive to IR. RZ (sstRZ) and XL (sstXL) are MAGE alleles. Df (Df(3R)Antp1) is a deficiency chromosome uncovering the MAGE locus. (C) Smc5 mutants are hypersensitive to IR. P5 (Smc5P{GSV1}GS3245) and P7 (Smc5P{GSV6}GS14577) are Smc5 alleles. Df (Df(3L)BSC418) is a deficiency chromosome uncovering the Smc5 locus.
Figure 6
Figure 6. Smc5/6 genes are not required for G2/M and S phase checkpoints induced by genotoxic agents.
(A) Wandering third instar larvae were irradiated with 40 Gy of ionizing radiation and the eye-antenna discs were dissected and fixed 15 minutes, 30 minutes, 1 hour or two hours after radiation, with discs from unirradiated larvae serving as controls. Representative images of PH3 staining for mitotic cells in eye-antenna discs from control (WT, FRT82B) and Smc6, (jnjR1/jnjX1) transheterozygous larvae are shown. (B) Eye-antenna discs from wandering third instar larvae were incubated with or without HU before adding BrdU to the incubation solution. Representative images of BrdU staining for cells in S phase in eye-antenna discs from control (WT, FRT82B), transheterozygous Smc6 (jnjR1/jnjX1) or transheterozygous MAGE (sstRZ/sstXL) eye-antenna discs are shown.
Figure 7
Figure 7. Caffeine-dependent genetic interaction of MAGE with ATM, ATR and Rad51(SpnA).
(A) Representative eye phenotypes of MAGE (EGUF/+; FRT82B sstRZ/FRT82B GMR-hid, loss of MAGE in eye cells), ey>ATMi (knockdown of ATM in eye cells), ey>ATMi;MAGE (EGUF/UAS-ATM-RNAi;FRT82B sstRZ/FRT82B GMR-hid, loss of MAGE and knockdown of ATM in eye cells) and ey>ATRi;MAGE (EGUF/UAS-ATR-RNAi;FRT82B sstRZ/FRT82B GMR-hid, loss of MAGE and knockdown of ATR in eye cells) flies that were reared on either standard media or media containing 2 mM caffeine. The EGUF system carrying the eyeless-Gal4 driver was used to drive the UAS-RNAi transgenes in the eye and also makes the eye homozygous for MAGE (sstRZ). Controls for the effects of each eyeless-driven RNAi alone were carried out for ATM and ATR resulting in wild type appearing eyes, but only the results of ATM RNAi are shown here as an example. (B) Representative eye phenotypes of MAGE knockdown (eyeless-Gal4/+;UAS-MAGE-RNAi/UAS-Dicer2, knockdown of MAGE in eye cells) and MAGE Rad51 double knockdown (eyeless-Gal4/UAS-SpnA-RNAi;UAS-MAGE-RNAi/UAS-Dicer2, knockdown of MAGE and Rad51 in eye cells) flies that were reared on either standard media or media containing 2 mM caffeine.
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