Figure 4. pch2∆ mutants display altered Hop1 occupancy and crossover MutLγ-dependence.

(A) Hop1 occupancy at insert loci, determined by chromatin immunoprecipitation and quantitative PCR. Top—cartoon of insert loci, showing the location of primer pairs used. Bottom—relative Hop1 occupancy, expressed as the average ratio of immunoprecipitate/input extract for both primer pairs (see Materials and methods for details). Values are the average of two independent experiments; error bars represent range. (B) VDE-initiated CO frequencies measured as in Figure 2C at HIS4 (top) and URA3 (bottom) in pch2∆ (solid diamonds), pch2∆ mlh3∆ (open diamonds), and pch2∆ mms4-md yen1 slx1 (half-filled diamonds) mutants. Crossovers from wild type (solid line), mlh3∆ (dotted line) and mms4-md yen1 slx1mutants (dashed line) from Figure 3 are shown for comparison. Values are from two independent experiments; error bars represent range. Representative Southern blots are in Figure 4—figure supplement 2. (C) Extent of CO reduction in mlh3∆ mutants, relative to corresponding MLH3 strains. (D) Extent of CO reduction in mms4-md yen1 slx1 (ssn) mutants, relative to corresponding MMS4 YEN1 SLX1 strains. For both (C) and (D), PCH2 genotype is indicated at the top; values are calculated as in Figure 3C.

DOI: http://dx.doi.org/10.7554/eLife.19669.010

Figure 4—figure supplement 1. Hop1 occupancy at non-insert loci, DSBs and NCOs in pch2∆ mutants.

(A) Hop1 occupancy at corresponding loci lacking inserts, determined as in Figure 4A. Occupancy at HIS4 is from strains with inserts at URA3, and vice versa. (B) DSBs and NCOs in inserts at HIS4, determined as in Figure 2B and C, respectively. Symbols are as in Figure 4B. (C) DSBs and NCOs in inserts at URA3, details as in panel B. Values are from two independent experiments; error bars represent range. Representative Southern blots are in Figure 4—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.19669.011

Figure 4—figure supplement 2. Southern blots of HindIII and HindIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom).

Gel labels are as in Figure 1; JM—joint molecule recombination intermediates. In the gel with HinDIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3, the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes.

DOI: http://dx.doi.org/10.7554/eLife.19669.012