The effect of mutations in <i>PMS1</i> on 2-nt in/del mispairs.

<p>Oligos were transformed into strains of the indicated genotypes and analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-g002" target="_blank">Figure 2</a>; the <i>msh2</i> results are those given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-g002" target="_blank">Figure 2</a>.</p

Effect of Mlh3 on 2-nt deletion mispairs.

<p>Oligos were transformed into strains of the indicated genotypes and analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-g002" target="_blank">Figure 2</a>. (Data for <i>msh2</i>, <i>msh6</i>, and <i>pms1-H888R</i> are from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-g002" target="_blank">Figures 2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-g003" target="_blank">3</a>.)</p

In/del Repair Ratios for 1 nt mispairs.

<p>Data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920.s003" target="_blank">Figure S3</a> were used to calculate Repair Ratios by using the ratio of revertants obtained in the absence of MMR (<i>msh2</i> strains) with the number of revertants in strains of the indicated genotype.</p

Effect of MMR on 2-nt in/del mismatches.

<p>The mean number of Lys+ revertants, with standard deviation, is shown for the indicated oligo and strain combination. The coloring is explained in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-g001" target="_blank">Figure 1</a> and oligo sequences are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920.s008" target="_blank">Table S4</a>. TrL1 and TrL2 refer to oligos with the sequence of the transcribed strand in Location 1 and 2, respectively. For the Tr oligos, annealing to the lagging strand occurs in strains with the Same orientation (Lag-s). The fewer transformants obtained for a given oligo and strain combination, the better the repair for the mismatch created by the oligo. Oligos creating insertion loops are transformed into <i>lys2ΔBgl</i> strains and oligos creating deletion loops are transformed into <i>lys2ΔA746</i> strains. As an example, all TrL1 oligos are essentially identical in sequence, with the exception that the “blue” oligo inserts a +GA loop, the “green” oligo inserts a +TC loop, and the “red” oligo causes a 2-nt −GA deletion loop in the template strand opposite the location of the + loops in the other two oligos. There is no active MMR in <i>msh2</i> strains, whereas <i>msh3</i> strains have MutSα present and <i>msh6</i> strains contain MutSβ.</p

Repair Ratios for 2-nt in/del mispairs.

<p>Data from Figures S1 and S2 were used to calculate Repair Ratios by using the ratio of revertants obtained in the absence of MMR (<i>msh2</i> strains) with the number of revertants in strains of the indicated genotypes. Only MutSβ is present in <i>msh6</i> strains and only MutSα is present in <i>msh3</i> strains.</p

Median Repair Ratios for 2-nt in/del mismatches.

<p>The median Repair Ratio for each genotype is calculated from the values with individual oligos in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-t001" target="_blank">Tables 1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-t005" target="_blank">5</a>, and S1.</p>a<p>The probability that the values for insertions were different from deletions was calculated using a Mann-Whitney rank sum test. N.S. indicates the two sets of values were not significantly different.</p

An assay for loop repair.

<p>(A) The initial strains used to construct the assay for in/del loop repair were a set of isogenic strains containing the <i>LYS2</i> gene replacing the <i>HIS4</i> gene near the <i>ARS306</i> origin of replication as shown above <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920-Kim1" target="_blank">[26]</a>. “Same” and “Opposite” refer to the orientation of the <i>LYS2</i> gene relative to the orientation of the original <i>HIS4</i> gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920-Kim1" target="_blank">[26]</a>. The wild-type <i>LYS2</i> sequences were subsequently replaced with sequences to create either the −1 frameshift allele <i>lys2ΔA746</i> or the +1 frameshift allele <i>lys2ΔBgl </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920-Kow1" target="_blank">[23]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920-Greene1" target="_blank">[25]</a>. (B) The −1 <i>lys2ΔA746</i> and the +1 <i>lys2ΔBgl</i> frameshift alleles can be reverted to wild-type by a compensating addition or deletion of nucleotides anywhere within an approximately 200-bp reversion window <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920-Kow1" target="_blank">[23]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920-Greene1" target="_blank">[25]</a>. Oligos with sequences corresponding to two different locations within the reversion window of the mutant alleles and ranging in size from 31–36 nt were used to produce Lys+ revertants (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920.s008" target="_blank">Table S4</a>). The colors indicated are those used in subsequent figures and also in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920.s008" target="_blank">Table S4</a>. The red and yellow oligos induce a 2-nt loss in <i>lys2ΔA746</i> strains and the blue and green oligos insert 2 nt into <i>lys2ΔBgl</i> strains. Single-stranded oligos are used for transformation, and can therefore have the sequence of either the transcribed (Tr) or non-transcribed strand (NTr). Oligos inducing 1-nt in/del mutations follow a similar color and naming scheme (see text for details). (C–F) Oligos transform by serving as primers for subsequent replication, on either the leading or lagging strands of replication. If the mismatch created by the oligo is not removed during replication, a reverting frameshift will result in the next round of replication. Additional nucleotides in the oligo will create a primer-strand loop and thus an insertion mutation; missing nucleotides in the oligo will create a loop on the template strand and thus lead to a deletion mutation. (C) and (D) indicate that the same oligo (an oligo with the sequence of the transcribed strand (Tr) in location 2 (L2) adding a sequence of TC) will anneal to the leading strand of replication in <i>lys2ΔBgl</i> strains of the <u>Opposite</u> orientation (TrL2-lead-o) or to the lagging strand in strains of the <u>Same</u> orientation (TrL2-lag-s). (E) and (F) show the same process for an oligo inducing a deletion of GA in <i>lys2ΔA746</i> strains by annealing on the leading strand in strains of the <u>Opposite</u> orientation (TrL2-lead-o) or on the lagging strand of strains in the <u>Same</u> orientation (TrL2-lag-s).</p

The effect of Mlh3 on Repair Ratios for 2-nt in/del mispairs.

<p>Repair Ratios were calculated as in previous tables.</p

Effect of MMR on 1-nt in/del mismatches.

<p>TrL1 Oligos were transformed into Same-orientation strains of the indicated genotypes and analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-g002" target="_blank">Figure 2</a> (TrL1-Lag-s). For 1-nt in/del mismatches, oligos creating insertion loops are transformed into <i>lys2ΔA746</i> strains and oligos creating deletion loops are transformed into <i>lys2ΔBgl</i> strains. Only MutSβ is present in <i>msh6</i> strains and only MutSα is present in <i>msh3</i> strains.</p

Median Repair Ratios for 1-nt in/del mismatches.

<p>The median Repair Ratio for each genotype is calculated from the values with individual oligos in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen-1003920-t003" target="_blank">Tables 3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003920#pgen.1003920.s006" target="_blank">S2</a>.</p>a<p>The probability that the values for insertions were different from deletions was calculated using a Mann-Whitney rank sum test. N.S. indicates the two sets of values were not significantly different.</p