Figure 6. Pif1 mutants inhibit break-induced replication.
(A) Top: Schematic of 30 kb repair template strain. The HO endonuclease is under control of a GAL promoter. In the presence of galactose, it is expressed, inducing a DSB on chromosome V. Sequences telomeric to the HO cut site are non-essential. Homology between the two incomplete URA3 fragments allows for BIR and subsequent telomere addition. Bottom: Frequencies of repair. The percentage of cells that are viable on galactose (compared to total cells plated on YPD) indicates the frequency of all repair events. The subset of those cells that grow on media lacking uracil (URA+) indicates the frequency of BIR events. (B) As in (A), but with a repair template 80 kb from the telomere. Bars represent mean +/- standard deviation (n = 4).
Figure 6—figure supplement 1. Mutants don’t affect non-homologous end joining or HO-induced DSBs.
(A) More-detailed schematic of chromosome V constructs being used in Figure 6. A gene that confers resistance to the drug cloNAT (natMX) is placed telomeric to the HO cut site (HOcs). Primers designed to flank the HOcs are shown as well. (B) Frequencies of cloNAT resistance in various genotypes in both 30- and 80 kb repair template strains. Viable colonies grown on galactose (Figure 6) were replica-plated to media containing cloNAT. Frequency is calculated by comparing cells that grew on cloNAT to total cells on YPD. Ability to grow on uracil and cloNAT resistance were mutually exclusive. Bars represent mean +/- standard deviation (n = 4). (C) Saturated cultures of the indicated genotype were diluted into media containing dextrose or galactose and allowed to divide for six hours. Genomic DNA was extracted and PCR was performed at the HO cut site (HOcs) or the RNH1 locus as a positive control. PCR products were loaded onto an agarose gel and stained with ethidium bromide.