ATP-dependent Chromatin Remodeling by the Saccharomyces cerevisiae Homologous Recombination Factor Rdh54

Abstract

Saccharomyces cerevisiae RDH54 is a key member of the evolutionarily conserved RAD52 epistasis group of genes needed for homologous recombination and DNA double strand break repair. The RDH54-encoded protein possesses a DNA translocase activity and functions together with the Rad51 recombinase in the D-loop reaction. By chromatin immunoprecipitation (ChIP), we show that Rdh54 is recruited, in a manner that is dependent on Rad51 and Rad52, to a site-specific DNA double strand break induced by the HO endonuclease. Because of its relatedness to Swi2/Snf2 chromatin remodelers, we have asked whether highly purified Rdh54 possesses chromatin-remodeling activity. Importantly, our results show that Rdh54 can mobilize a mononucleosome along DNA and render nucleosomal DNA accessible to a restriction enzyme, indicative of a chromatin-remodeling function. Moreover, Rdh54 co-operates with Rad51 in the utilization of naked or chromatinized DNA as template for D-loop formation. We also provide evidence for a strict dependence of the chromatin-remodeling attributes of Rdh54 on its ATPase activity and N-terminal domain. Interestingly, an N-terminal deletion mutant (rdh54⌬102) is unable to promote Rad51-mediated D-loop formation with a chromatinized template, while retaining substantial activity with naked DNA. These features of Rdh54 suggest a role of this protein factor in chromatin rearrangement during DNA recombination and repair.The genomic DNA in eukaryotic cells is organized into chromatin, which harbors repeated nucleosome units each comprising an octamer of four histone proteins, H2A, H2B, H3, and H4, and 147 bp of DNA (1, 2). The folding of DNA on the surface of histone proteins within the nucleosome inevitably poses an accessibility problem during various DNA transactions, including DNA replication, recombination, and repair.One mechanism to efficiently overcome this structural hindrance is by an ATP hydrolysis-dependent chromatin-remodeling process, such that remodeling proteins render DNA more accessible by weakening DNA:histone contacts, sliding nucleosomes along DNA, or removing H2A-H2B dimers from the nucleosome (3-5).We are interested in delineating the mechanism of homologous recombination (HR) 2 that is mediated by proteins of the RAD52 epistasis group, including how the HR machinery negotiates the constraints posed by chromatin structure. Regarding the latter, of special interest are two key members of the RAD52 epistasis group, Rad54 and Rdh54 proteins. These HR factors play multifaceted roles in HR (6 -8), and they belong to the Swi2/Snf2 protein superfamily, members of which are well known ATP hydrolysis-dependent chromatin remodelers (9, 10). Rad54 and Rdh54 both possess a DNA-dependent ATPase activity and a dsDNA translocase function that is fueled by ATP hydrolysis (8,(11)(12)(13)(14)(15). The DNA translocase activity of these Swi2/Snf2-like HR factors can modify DNA topology (11-13), enhance D-loop formation by the Rad51 recombinase (11, 16), accelerate the rate at which DNA strands are exchanged duri...

Chromatin dynamics coupled to DNA repair

“…115 These results are consistent with the ability of Rad54 to displace Rad51 from DNA in an ATP-dependent reaction. 116,117 In contrast to previous reports, Shina and Peterson 115 suggest that the chromatin remodeling activity of SWI/SNF does not generally enhance homology search in chromatin donors. The role of SWI/SNF in HR repair may be restricted to situations in which the donor locus is embedded in a highly condensed, heterochromatic chromatin structure.…”

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

“…At various times after HO induction, an aliquot of the yeast cultures was processed for ChIP, using IgG Sepharose beads to precipitate TAP-tagged Mph1 and associated DNA. Radioactive PCR with the appropriate primer sets was then used to amplify the target sequences: MAT Z (the invading strand) and HMLa (the recombination donor) (Wolner et al 2003;Kwon et al 2008). As shown in Figure 3B, Mph1 is recruited to the MAT Z region in the donorless strain (JKM179) after DSB induction.…”

“…The PCR reaction mixtures were resolved in a 6% nondenaturing polyacrylamide gel run in TBE buffer (40 mM Tris borate at pH 7.4, 0.5 mM EDTA) and quantified by phosphorimaging analysis. The MAT Z or HMLa signal at each time point was divided by the corresponding PHO5 signal and normalized to the 0-h time-point signal, as described (Wolner et al 2003;Kwon et al 2008). …”

Regulation of DNA Pairing in Homologous Recombination

“…These proteins are also able to process or dissociate DNA structures, such as the D-loop and branched DNAs including the Holliday junction and to remove Rad51 from dsDNA via their DNA translocase activity (Solinger et al 2002;Bugreev et al 2006Bugreev et al , 2007. Consistent with their Swi2/Snf2 relatedness, a chromatin-remodeling activity has been found in these proteins (Alexeev et al 2003;Kwon et al 2008).…”