Sequence divergence impedes crossover more than noncrossover events during mitotic gap repair in yeast - PubMed (original) (raw)

Sequence divergence impedes crossover more than noncrossover events during mitotic gap repair in yeast

Caroline Welz-Voegele et al. Genetics. 2008 Jul.

Abstract

Homologous recombination between dispersed repeated sequences is important in shaping eukaryotic genome structure, and such ectopic interactions are affected by repeat size and sequence identity. A transformation-based, gap-repair assay was used to examine the effect of 2% sequence divergence on the efficiency of mitotic double-strand break repair templated by chromosomal sequences in yeast. Because the repaired plasmid could either remain autonomous or integrate into the genome, the effect of sequence divergence on the crossover-noncrossover (CO-NCO) outcome was also examined. Finally, proteins important for regulating the CO-NCO outcome and for enforcing identity requirements during recombination were examined by transforming appropriate mutant strains. Results demonstrate that the basic CO-NCO outcome is regulated by the Rad1-Rad10 endonuclease and the Sgs1 and Srs2 helicases, that sequence divergence impedes CO to a much greater extent than NCO events, that an intact mismatch repair system is required for the discriminating identical and nonidentical repair templates, and that the Sgs1 and Srs2 helicases play additional, antirecombination roles when the interacting sequences are not identical.

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Figures

F<sc>igure</sc> 1.—

Figure 1.—

The gap-repair assay. The lengths of homology that flank the gap on the 5′ and 3′ sides are ∼600 bp and 160 bp, respectively. See text for further explanation of the system.

F<sc>igure</sc> 2.—

Figure 2.—

The genetic control of homologous gap repair. The total gap-repair efficiency using an identical chromosomal template (the _his3-0,Δ_3′ allele) in each strain background was measured as the ratio of His+ to Leu+ transformants. These ratios were then normalized to that obtained in the WT strain. The open and shaded areas within each bar correspond to the NCO and CO efficiencies, respectively, and were obtained by multiplying the total (normalized) efficiency by the proportion of the relevant event. The standard deviation of the total efficiency in each strain is indicated.

F<sc>igure</sc> 3.—

Figure 3.—

Homologous and homeologous gap repair (HGR and HeGR, respectively) in a WT strain. Strains containing a homologous or homeologous repair template (the _his3-0,Δ_3′ or _his3-18,Δ_3′ allele, respectively) were transformed with the gapped plasmid and the total (NCO + CO) repair efficiency was measured as the ratio of His+ to Leu+ colonies. These ratios were normalized to that obtained with the homologous, 100%-identical substrates; the standard deviations of the total efficiencies are indicated. NCO and CO efficiencies were obtained by multiplying the total (normalized) efficiency by the proportion of the relevant event. Open and shaded bars correspond to HGR and HeGR efficiencies, respectively.

F<sc>igure</sc> 4.—

Figure 4.—

Effect of sequence divergence on gap repair in different genetic backgrounds. The HeGR efficiency (His+/Leu+) ratio was normalized to the HGR efficiency obtained in the same genetic background. The NCO and CO efficiencies were determined by multiplying the total repair efficiency by the proportion of the relevant event and these efficiencies were used to calculate the corresponding HeGR/HGR ratio. If sequence divergence has no effect on repair efficiency, then HeGR/HGR = 1. An HeGR/HRG ratio <1 indicates that sequence divergence inhibits repair; the smaller the ratio, the greater the inhibition. The double arrowheads to the right indicate the magnitude of the inhibition in the WT background.

F<sc>igure</sc> 5.—

Figure 5.—

Models for recombinational repair of a gapped plasmid using chromosomal DNA as a template. Plasmid and chromosomal DNA are indicated as black and red lines, respectively. Arrowheads represent 3′ ends, and dotted lines correspond to newly-synthesized DNA. The colors of the dotted lines correspond to that of the template. Heteroduplex DNA forms adjacent to the original gap and is depicted as paired black and red lines. Details of the models are given in the text.

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