Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast - PubMed (original) (raw)

Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast

A Datta et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Sequence divergence acts as a potent barrier to homologous recombination; much of this barrier derives from an antirecombination activity exerted by mismatch repair proteins. An inverted repeat assay system with recombination substrates ranging in identity from 74% to 100% has been used to define the relationship between sequence divergence and the rate of mitotic crossing-over in yeast. To elucidate the role of the mismatch repair machinery in regulating recombination between mismatched substrates, we performed experiments in both wild-type and mismatch repair defective strains. We find that a single mismatch is sufficient to inhibit recombination between otherwise identical sequences, and that this inhibition is dependent on the mismatch repair system. Additional mismatches have a cumulative negative effect on the recombination rate. With sequence divergence of up to approximately 10%, the inhibitory effect of mismatches results mainly from antirecombination activity of the mismatch repair system. With greater levels of divergence, recombination is inefficient even in the absence of mismatch repair activity. In both wild-type and mismatch repair defective strains, an approximate log-linear relationship is observed between the recombination rate and the level of sequence divergence.

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Figures

Figure 1

Figure 1

Construction of inverted repeat substrates. The pGAL-HIS3::intron construct contained on plasmid pSR266 is shown at the top. Open boxes correspond to HIS3 sequences, solid boxes to artificial intron sequences, and cross-hatched boxes to cβ sequences; boxes are not to scale. Only those restriction sites relevant to the constructions are shown: Sal, _Sal_I; Sma, _Sma_I; Bam, _Bam_HI; Spe, _Spe_I; Not, _Not_I; Nae, _Nae_I; Bgl, _Bgl_II.

Figure 2

Figure 2

Alignments of recombination substrates derived from cβ cDNA sequences. All sequences are approximately 350 bp. Each potential mismatch between a given pair of sequences is indicated by a vertical line.

Figure 3

Figure 3

Recombination rate versus % sequence divergence. All the data in Table 1 are graphed. (Inset) Data for the 0–4 mismatch substrates only. Open and solid circles correspond to data from MMR+ and MMR− strains, respectively. The curves were derived using the model described in the text. Nonlinear curves were fit using the simplex method of

systat

5.0 (Macintosh). Different starting conditions for the nonlinear curve fitting program produced nearly 5-fold variations in the fitted values of _R_0 and _P_0, which were statistically indistinguishable from each other; fitted values of the other parameters remained approximately constant with a range <1%.

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