Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers

Abstract

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.

Figure 1.—

Vectors for gene targeting in Drosophila. Schematic maps of pP{Target} and pP{TargetB} are shown. Both vectors carry a mini-white marker gene (shaded arrow), FRTs (stippled triangles), an I-CreI endonuclease recognition sequence (X), and P-element ends for transposition (solid arrows). The two vectors differ in size and in restriction sites available for cloning.

Figure 2.—

Targeting of Ercc1. (A) Genes from the Ercc1 genomic region are shown as solid arrows (indicating direction of transcription) on a solid line, and genes on the targeting DNA are shown as open arrows or boxes on a shaded line. The targeting DNA is shown after excision by FLP recombinase and cutting by I-SceI endonuclease, with an asterisk marking the site of the double-strand break. This targeting fragment is drawn to show alignment of sequences with homology between the targeting DNA and the genomic DNA. The XhoI site introduced into Ercc1 is indicated by an X. The mini-white marker gene and the FRT and I-CreI sites are as in Figure 1. (B) The predicted product of ends-in integration with sequences derived from the targeting DNA in open symbols and chromosomal sequences as solid symbols. The region deleted in the integration that we recovered is indicated. (C) Predicted products from reduction of the tandem duplication after cutting with I-CreI and repair by single-strand annealing. One product is completely wild type (top), and the other carries Ercc1^X and the adjacent deletion of Ciao1 (bottom). (D) The structure of the mutation used in these studies. It is equivalent to the targeted integration depicted in B, except that most of mini-white and one copy of Ercc1 have been deleted, and the remaining copy of Ercc1 carries the XhoI mutation.

Figure 3.—

Molecular analysis of tandem duplication and Ercc1^X reduction. Allele-specific PCR was performed using either an Ercc1-specific primer (+) or a Ercc1^X-specific primer (X) and a reverse primer complementary to sequence outside of the targeting sequence. PCR reactions were run on a standard agarose gel for analysis. The expected 2.4-kb product is seen with the “+” primer in wild type; however, the expected 2.4-kb product is missing with the X primer in Ercc1^X and instead a 1.2-kb product is seen. A 800-bp product is seen with the X primer in the duplication, confirming the presence of the 1.6-kb deletion.

Figure 4.—

Sensitivity to killing by UV light of Ercc1^X and mei-9 mutants. Percentage survival, relative to wild-type controls, after exposure of larvae to 500–1000 erg/mm² (1 erg/mm² = 0.1 J/m²) of UV light is shown for Ercc1^X homozygous and hemizygous mutants and mei-9^A2 mutants. Bars indicate standard deviations.

Figure 5.—

Expression of MEI-9 in ovaries. Ovarian proteins were separated on a polyacrylamide gel, transferred to PVDF membrane, and detected with polyclonal anti-MEI-9 serum. Genotypes of ovaries are (1) wild type, (2) mei-9^A2, (3) zygotic Ercc1^X, (4) maternal/zygotic Ercc1^X, and (5) P{WUF9} in maternal/zygotic Ercc1^X. MEI-9 is indicated by a solid arrowhead (M_r = 125 kDa). The antiserum also detects unknown proteins of lower molecular weight; one of these is included as a loading control. Flag-tagged MEI-9 protein (open arrowhead) produced from the P{WUF9} transgene migrates slightly faster than untagged MEI-9 (M_r = 113 kDa). The mei-9 cDNA used to make this construct lacks 35 nonessential amino acids at the N terminus (see materials and methods for details).

Figure 6.—

Yeast two-hybrid assay with ERCC1, MUS312, and MEI-9. ERCC1 was expressed in yeast as a fusion protein with the Gal4 DNA-binding domain (GBD) in a vector that either did (2, 4, 6) or did not (1, 3, 5) also express MEI-9. Yeast were transformed with an empty Gal4 activation domain (GAD) vector (1, 2), a vector expressing GAD-MEI-9 fusion protein (3, 4), or GAD-MUS312 fusion protein (5, 6). Growth on the −HIS dropout media shown indicates an interaction between the GBD and GAD fusion proteins.