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. 1999 Mar 16;96(6):2964-9.
doi: 10.1073/pnas.96.6.2964.

Microsatellite instability in Drosophila spellchecker1 (MutS homolog) mutants

C Flores  1 , W Engels
Affiliations

Microsatellite instability in Drosophila spellchecker1 (MutS homolog) mutants

C Flores et al. Proc Natl Acad Sci U S A. .

Abstract

We have cloned a mutS homolog from Drosophila melanogaster called spellchecker1 (spel1) and have constructed spel1 mutant flies. MutS proteins promote the correction of DNA mismatches and serve important roles in DNA replication, recombination, and repair. The spel1 gene belongs to a subfamily of mutS first characterized by the MSH2 gene of yeast and which also includes hMSH2, one of the two major hereditary nonpolyposis colon cancer loci of humans. Like msh2 mutants in other species, we find that flies lacking the spel1 gene suffer a highly increased rate of instability in long runs of dinucleotide repeats when analyzed after 10-12 fly generations. Using a new assay, we have also discovered that mutations in spel1 decrease the stability of a dinucleotide repeat when it is copied into the site of a double-strand break during gene conversion. Contrary to the case in mammalian cells, spel1 deficiency does not affect tolerance of flies to a methylating agent nor does it affect resistance to gamma-irradiation.

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Figures

Figure 1
Figure 1
Gene and transcript map of spel1. (A) The genomic region around spel1 is shown. Deletion-containing chromosomes used are displayed as black arrows representing the retained regions; on the opposite side, beyond the parentheses, blank areas indicate sequences that are absent. The regions of dashed line indicate the extent of uncertainty in the deletion end-points. The l(2)35Aa is a genetically defined locus known from ethyl methylsulfonate-induced mutations and was mapped by the GW7 and A400 deficiencies as shown. The solid and open portions represent the minimal and maximal extent of this locus. The neighboring genes GalNAc-T and ppk are localized from the DNA sequence. The numbering system is the one presented in Davis et al. (45). (B) The spel1 gene is enlarged with 5′ ends and sites of 3′ poly(A) in cDNAs indicated. Directly below the gene, the black bar represents the PCR fragment that was used as the initial probe. The PstI restriction site was used to test the homogeneity of the product of degenerate PCR. Examples of full-length or nearly full-length transcripts are shown below (one with the fifth intron retained and one completely spliced).
Figure 2
Figure 2
Microsatellite instability during double-strand break repair. (A) Genotype of females in which a microsatellite template is copied into the site of a double-strand break. One of the X chromosomes carries whd, a P element insertion in the white locus that causes a double-strand break on excision (47). Its allele, wA87.2, was derived from whd as described (48). In this allele, the interior of the P element is replaced by a (TA)23 microsatellite. Only the outermost 17 bp of the P element remain at each end. Thus, double-strand breaks produced by excision at whd can be repaired by copying in the microsatellite sequence from the homolog. Chromosome 2 of these females is null for spel1 via the combination of overlapping deletions and has the rescuing GalNAc-T transgene in P{CaGal}, as described above. Finally, a source of P transposase is provided on chromosome 3 (49). (B) Sons from these females whose X chromosome was derived from the whd parent were selected using the yellow and split loci as markers (37). Among 4,377 such males tested individually by using PCR, we found that 40% had acquired the wA87.2 allele, as judged by agarose gel electrophoresis. This frequency is consistent with previous results (48). From these, we selected no more than one son from each female to analyze with the high-resolution genescan method as described above. That way, all gene conversions can be considered independent events. Of 276 such sons from spel1-null mothers, we found eight size changes: one 22-base expansion, four 2-base expansions, and three 2-base contractions. The controls (Ctl) were sons from females similar to those in A except that one or both of the spel1− deletions is replaced with a spel1+ chromosome. Among 541 sons in this group, there were three size changes, a 2-base expansion and contractions of 4 and 10 bp.
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