Abstract
The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic interhomologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. Here we describe an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, we demonstrate that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. We furnish evidence that the Rad51 binding domain resides within the N terminus of Rdh54. Accordingly, N-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54Delta mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54.Rad51 complex formation. The Rdh54 expression and purification procedures described here should facilitate the functional dissection of this DNA recombination/repair factor.
Highlights
The genetic requirement of Homologous recombination (HR) was initially defined in the budding yeast Saccharomyces cerevisiae
We examined haploid and diploid rdh54K352R strains for their sensitivity to methyl methanesulfonate (MMS), which induces DNA damage that is repaired by HR
Genetic studies conducted in several laboratories have shown a role of the RDH54 gene in mitotic and meiotic HR, in those events that involve homologous chromosomes, i.e. interhomologue recombination [9, 10]
Summary
Plasmids—DNA fragments encoding full-length and N-terminally truncated variants of Rdh were prepared by the PCR reaction and cloned into the NcoI and XhoI site of the pET32a vector (Novagen) to add thioredoxin and His tags to the N terminus of these proteins. ATPase Assay—The indicated amounts of Rdh and truncated variants were incubated at 37 °C in 10 l of buffer D (35 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 3 mM MgCl2, and 50 mM KCl) containing 100 g/ml bovine serum albumin, 25 Ci/mol of [␥-32P]ATP at the final concentration of 1 mM, and pBluescript II SK DNA (23 M base pairs). DNA Mobility Shift Assay—The 32P-labeled 80-mer dsDNA (30 nM) was incubated for 5 min at 37 °C with the indicated amounts of Rdh and truncated variants in 10 l of buffer D with or without 2 mM ATP and an ATP-regenerating system consisting of 20 mM creatine phosphate and 30 g/ml creatine kinase. The various supernatants and SDS eluates (8 l each) were analyzed by SDS-PAGE and Coomassie Blue staining to determine their content of proteins and by agarose gel electrophoresis in TAE buffer followed by ethidium bromide staining to reveal the pBluescript dsDNA trap
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
