Rdh54 prevents Rad54 from creating accessible 3’ end at D-loop intermediates.

(A). Cartoon diagram illustrating the experiment to monitor the 3’ end accessibility by using Klenow (exo-) to extend the nascent D-loop. The recipient DNA used in this experiment was 21 nt of homologous ssDNA and 36 bp of duplexed non-homologous DNA (B). Representative gel illustrating D-loop extension for Rad51 alone, Rad51+Rad54 (30 nM), Rad51+Rdh54 (30 nM), and Rad51+Rad54 (30 nM) +Rdh54 (30 nM). (C). Quantification of extended D-loop fraction. The error bars represent the standard deviation for three independent experiments.</p

Gene conversion outcomes are unaffected in N-terminal chimeric Rad54 and Rdh54.

(A) Graph representing the red/white/sectored colony outcomes for all strains used in this paper. Strains are labeled in the figure. The bars and error bars represent the mean and standard deviation of at least three independent experiments. (TIF)</p

Comparison of N-terminal Chimeras for solid red and solid white colonies.

(A). Quantification of sectored red colonies in which both sister chromatids have undergone STGC for BIR or BIR like, CO, and NCO for WT, rdh54Δ/rdh54Δ, rad54Nrdh54/ rad54Nrdh54, and rdh54Nrad54/ rdh54Nrad54 strains. The WT and rdh54Δ strains are reproduced from S1 Fig. The columns represent the mean, and the error bars represent the standard deviation of at least 3 independent experiments. (B). Quantification of sectored white colonies in which both sister chromatid has undergone LTGC for BIR or BIR like, CO, and NCO for WT, rdh54Δ/rdh54Δ, rad54Nrdh54/ rad54Nrdh54, and rdh54Nrad54/ rdh54Nrad54 strains. The WT and rdh54Δ strains are reproduced from S1 Fig. The columns represent the mean, and the error bars represent the standard deviation of at least 3 independent experiments. (TIF)</p

Rdh54 kinetically effects D-loop maturation on longer DNA templates.

(A). Schematic diagram illustrating the longer fluorescently recipient DNA used in this experiments. The recipient DNA used in this experiment was 65 nt of homologous ssDNA and 25 bp of non-homologous dsDNA (B). Representative agarose gel representing a D-loop extension assay with Rad54 (30 nM) at 2, 5, 10, 20, 30, and 45 minutes. (C). Representative agarose gel representing D-loop extension assay with Rad54 (30 nM) + Rdh54 (30 nM) at 2, 5, 10, 20, 30, and 45 minutes. (D). Representative agarose gel representing D-loop extension assay with Rad54 (30 nM) + Rdh54K318R (30 nM) at 2, 5, 10, 20, 30, and 45 minutes. (E). Bar graph representing quantification of the fraction of D-loops that are extended at 10, 20, 30, and 45 min. The error bars represent the mean and standard deviation of 4–10 independent experiments. (F). A plot illustrating the reaction half-time generated from a single-phase association fit of the percentage D-loop extension. The crossbars represent the mean, and the error bars represent the upper and lower limit of the half-time based on the fit of the data.</p

Rad51 binding site switching of Rdh54 and Rad54 alters 3’ end accessibility and post gene conversion outcomes.

(A). Representative gel image of experiments designed to test how efficiently Rdh54, Rad54NRdh54, and Rdh54NRad54 interfere with Rad54 mediated 3’ end accessibility to Klenow fragment. The recipient DNA in this experiment was 21 nt of homologous ssDNA and 36 bp of non-homologous DNA (B). Quantification of extended D-loops for samples with Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54. The data points and error bars represent the mean and standard deviation of at least three independent experiments. (C). Graph representing the plating efficiency (+Gal/-Gal) for WT-KanMX, rad54nrdh54, and rdh54nRad54 yeast strains. The data represents the mean and standard deviation for four independent experiments. (D). Bar graphs representing the NCO (left), CO (middle), and BIR (right) outcomes for WT-KanMX, rad54nrdh54, and rdh54nrad54. The data represent the mean and standard deviation of at least four independent experiments. The WT and rdh54Δ results are reproduced from previous figures for comparison purposes.</p

Analysis of RDH54 in post gene conversion recombination.

(A). Schematic diagram illustrating assay used to monitor recombination between homologous chromosomes during a double strand DNA break. The reporter is located on chromosome XV and only one homolog has an active I-SceI site. The inactivating mutations ade2-n is shown by a red line in the diagram. If Short tract gene conversion occurs (STGC) this mutation is lost and the ADE2 becomes active. (B). Yeast colonies plated after ± induction of I-SceI using galactose. (C). Schematic diagram illustrating the potential outcomes for DNA repair via homologous recombination. In the first step gene conversion can occur through long tract or short tract DNA repair. Following cellular division, non-crossover, crossover, and break induced replication (non-reciprocal exchange) outcomes can be determined for each event. (D). Cartoon illustration of HR outcomes that can be determined from this assay. The red lines indicate DNA extension.</p

Rdh54 does not alter Rad54 ATPase functions at physiological ratios.

(A). ATPase assay illustrating the fraction of Rad54 ATP hydrolysis in the presence and absence of Rad51 with and without Rdh54K318R. The bars represent the mean and standard deviation of three independent experiments. (B). Quantification of the number of fluorescently labeled Rad54 (N = 100) and Rdh54 (N = 100) molecules in yeast nucleus. The line and error bars represent the mean and 95% confidence interval of the data. The red number above the data is the mean molecules per cell. (TIF)</p

All red/white sectored outcomes CO/NCO/BIR.

(PDF)</p

Rdh54 inhibits Rad54 mediated D-loop turnover.

(A). Cartoon diagram illustrating the experimental design for monitoring Rad54 mediated D-loop maturation. The recipient DNA in this experiment is 21 nt of homologous DNA with 36 bp of non-homologous duplexed DNA (B). Representative agarose gels illustrating D-loop turnover for Rad51 alone, Rad51+Rad54 (30 nM), Rad51+Rad54+Rdh54 (10 nM), Rad51+Rad54+Rdh54 (30 nM), Rad51+Rad54+Rdh54 (90 nM), Rad51+Rdh54 (30 nM). (C). Quantification of D-loop turnover experiment as a Fraction of D-loop. The error bars represent the standard deviation of three independent experiments. (D). Normalized quantification of D-loop turnover. Experiments were normalized by setting the maximum fraction D-loop (10 min) to one and normalizing the other time points to that value. The error bars represent the standard deviation of three independent experiments. (E). Graphical depiction of fits of the D-loop turnover phase (10–45 min) of the reaction. The rates are represented from the slope of the best fit line for 0, 3:1, 1:1, and 1:3 molar ratios of Rad54:Rdh54.</p

Loss of <i>RDH54</i> results in an increase in BIR outcomes.

(A). Graph representing the plating efficiency of WT, RDH54-KanMX/RDH54-KanMX, RDH54/rdh54Δ, rdh54Δ/rdh54Δ, rdh54K318R/rdh54K318R, and rad54Δ/rad54Δ. Crossbar and error bars represent the mean and standard deviation of independent measurements. (B). Graphical representation of the fraction of cells that are red, white, or sectored after DNA double strand break repair. Strains represented here are WT, RDH54/rdh54Δ, rdh54Δ/rdh54Δ, RDH54-KanMX/RDH54-KanMX, rdh54K318R/rdh54K318R. The column and the error bars represent the mean and standard deviation of at least three independent experiments. (C). Graph representing the percentage of non-crossover outcomes for WT, RDH54-KanMX/RDH54-KanMX, RDH54/rdh54Δ, rdh54Δ/rdh54Δ, rdh54K318R/rdh54K318R. Measurements represent the mean and standard deviation of four independent experiments. (D). Graph representing the percentage of crossover outcomes for WT, RDH54-KanMX/RDH54-KanMX, RDH54/rdh54Δ, rdh54Δ/rdh54Δ, rdh54K318R/rdh54K318R. Measurements represent the mean and standard deviation of four independent experiments. (E). Graph representing the percentage of Break induced replication (BIR) outcomes for WT, RDH54-KanMX/RDH54-KanMX, RDH54/rdh54Δ, rdh54Δ/rdh54Δ, rdh54K318R/rdh54K318R. Measurements represent the mean and standard deviation of at least four independent experiments.</p