Characterization of the Interaction between the Saccharomyces cerevisiae Rad51 Recombinase and the DNA Translocase Rdh54

Frequent Interchromosomal Template Switches during Gene Conversion in S. cerevisiae

“…Dmc1 is not expressed in mitotic cells, but Rdh54 may adopt a similar role with Rad51 in these unusual circumstances. Indeed our analysis showed that the rdh54 -Δ 75 mutation which eliminates its interaction with Rad51 (Santa Maria et al, 2013) behaves as a null mutation (Fig 5C). …”

“…Rdh54 is ATP-dependent DNA translocase and interacts with Rad51 protein through its N-terminus (Chi et al, 2006; Santa Maria et al, 2013). To elucidate if translocase activity or interaction with Rad51 is required for template switch, we transformed rdh54 Δ strain with plasmids carrying full-length RDH54 (pHK489), rdh54 lacking first 75 amino acids required for interaction with Rad51 (pHK549) or rdh54-K318R (pRB101), all expressed from their native promoter (Santa Maria et al, 2013).…”

“…To elucidate if translocase activity or interaction with Rad51 is required for template switch, we transformed rdh54 Δ strain with plasmids carrying full-length RDH54 (pHK489), rdh54 lacking first 75 amino acids required for interaction with Rad51 (pHK549) or rdh54-K318R (pRB101), all expressed from their native promoter (Santa Maria et al, 2013). Full length RDH54 restored ICTS to the wild-type level, while neither rdh54 -Δ 75 nor rdh54-K318R constructs were able to rescue the rdh54 phenotype (Fig 5C and Table S2).…”

“…As constitutive overexpression of RAD51 is deleterious in a rdh54 Δ strain ((Santa Maria et al, 2013) and data not shown), we transformed rdh54 Δ with galactose-inducible RAD51 on a multicopy plasmid pHK317(3) (Santa Maria et al, 2013) and simultaneously induced a DSB and RAD51 overexpression for five hours. Transient Rad51 overproduction did not affect the rate of ICTS in the rdh54 Δ strain (Table S2), but significantly reduced its survival to 47% compared to the rdh54 Δ strain transformed with an empty vector (Table S4).…”

“…To express RDH54 , rdh54 -Δ 75 or rdh54-K318R from their native promoters, previously described plasmids pHK489, pHK549 and pRB101 were used (Santa Maria et al, 2013). To overexpress RDH54 , a full length RDH54 under its native promoter was PCR-amplified using pHK489 (Santa Maria et al, 2013) as a template and then inserted into the 2µ YEp13- LEU2 vector (Broach et al, 1979) by in vivo recombination creating pOT23.…”