Reduction of stability of arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function

Abstract

Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers. To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines. AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility. To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants. Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ([+2], [-2], [+4], or [-4] bp). For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608. Two of four AtMSH2-RNAi plants showed similar microsatellite instability. In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested. This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity. We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots. Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats. Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue.

Figure 1.

Structure of T-DNA insertion in At-MSH2. A, Sequences of PCR products generated with gene-specific and T-DNA-specific primers were used to deduce the structure of the disrupted AtMSH2 in the line SALK_002708. A single insertion of pROK2 T-DNA at positions 2,714 and 4,225 caused deletion of exons 8 to 12 and portions of exons 7 and 13 (gray boxes) in this line. Sequences of junction regions are below. Capital letters indicate AtMSH2 and lowercase letters indicate the insertion, beginning 2 bp downstream of the left border at the exon 7 junction and preceded by approximately 150 bp of rearranged sequence following the right border at the exon 13 junction. B, DNA blot of EcoRV-digested wild-type and T-DNA insertion homozygotes probed with a radiolabeled At-MSH2 fragment (dashed line).

Figure 2.

PCR amplification of DNA containing each of nine different microsatellites from each progeny plant with one of each locus-specific primer pairs, analysis of product mixtures by capillary electrophoresis, detection of fluorescent products, and generation of electropherograms were as described under “Materials and Methods.” A, Electropherograms showing all PCR products; one of each primer pair was labeled with FAM or HEX fluorescent dye. B, Representative electropherogram patterns for locus NGA1107. Apparent absolute size of primary wild-type (wt) product band is 317 bp. Shown also are products where both alleles showed an increase of one repeat unit, increasing apparent product length to 319 bp (+2), where one allele maintained wild-type length and one showed a loss of one repeat [wt & (-2)], and where one allele remained wild type and one showed a shift of two repeats, decreasing apparent length to 313 bp [wt & (-4)]. Arrows indicate position of 317-bp (wild-type) product. C, Patterns for reconstruction mixtures. DNA from plants homozygous (pure) for alleles at locus NGA1107 encoding 313-bp (black arrow) or 315-bp (gray arrow) products or mixtures of the two at indicated ratios (concentrations of DNAs measured before PCR by staining with PicoGreen [Molecular Probes, Eugene, OR]) were analyzed as described above. Note that the primary criterion for scoring a pattern as reflecting a mixture of two different length products—second peak from right greater than a significantly large first peak—is met only for 1:1 and 2:1 mixtures.

Figure 3.

Frequency (%) of repeat length-shifted alleles in endogenous microsatellite loci. Frequencies were calculated by dividing numbers of unique length shifts (A) or total numbers of all alleles showing non-parental lengths (B) in the progeny of two AtMSH2^+/+ plants or of three AtMSH2^-/- plants or of four individual AtMSH2(RNAi) plants by the total number of alleles tested in each group. Data represent sums for all nine loci analyzed or for the six loci with ≥25 repeat units. No length shifts were detected in progeny of two plants transformed with the empty binary vector pFGC5941.