There are 17 Snf2/Swi2-like proteins in budding yeast [12
], but ScRdh54 is the paralog that is evolutionarily most similar to Rad54 by functional and phylogenetic criteria, and we therefore treat ScRdh54 as the sole Rad54 paralog in the stricter sense. Both proteins appear to perform partially overlapping roles in HR as discussed in more detail below (). Similarly in humans, where several dozen Snf2/Swi2-like proteins exist, hRAD54B represents the only hRAD54 paralog clearly involved in the core mechanism of HR () [12
]. Other Snf2/Swi2-like proteins, such as Rad5 (human HLTF and SHPRH), Rad16, and Rad26 (human CS-B) are involved in different DNA repair pathways. In addition, the classical ATP-dependent chromatin remodeling factors and other Snf2/Swi2-like factors function in other aspects of the DNA damage response and transcriptional control (for review see [7
Budding yeast cells deficient for the Rad54 paralog ScRdh54 display very mild phenotypes compared to mutants in RAD54
. Mutants in RDH54
exhibit no MMS or IR sensitivity, somewhat increased levels of intrachromosomal recombination compared to wild-type, and only very slightly reduced interchromosomal recombination [44
]. Mitotic recombination is reduced in the diploid mutant strains but no reduction is observed in haploids [87
]. The meiotic phenotype of rdh54
cells is also rather mild with 82% spore viability compared to 53% for rad54
cells. However, the rad54 rdh54
double mutant is completely defective in meiosis, suggesting that both proteins perform partially overlapping functions in meiosis [44
]. It has been suggested that ScRdh54 preferentially functions in HR between homologs, possibly through its preferential interaction with Dmc1 (see ), the meiosis-specific Rad51 paralog that is required for meiotic interhomolog recombination [5
], whereas Rad54 is envisioned to primarily function in DNA repair involving the sister chromatid through its association with Rad51 [44
]. Further insights into the differential roles of ScRad54 and ScRdh54 will likely shed light on the mechanisms governing interhomolog events during meiotic recombination.
In somatic yeast cells, Rdh54 is recruited to a DSB by Rad51 and Rad52, as demonstrated in ChIP experiments [90
]. This result is rather surprising, given that ScRdh54-deficient cells are not sensitive to this type of DNA damage. However, ScRdh54 has also been implicated in signaling from DSBs, specifically during adaptation from DSB-induced cell cycle arrest in cells with a non-repairable DSB [91
]. While wild type cells eventually adapt after prolonged arrest and resume the cell cycle even in the presence of a DSB, rdh54
cells remain terminally arrested. It is possible that the localization of ScRdh54 and its recruitment by Rad51 and Rad52 is related to this signaling function [92
]. The mechanisms involved remain to be determined. In vivo
, ScRdh54 localizes to kinetochores, but the functional significance is unclear [84
]. In cells lacking ScRdh54, ScRad54 appears to take the place of ScRdh54 at kinetochores [84
], suggesting that this localization is not related to the ScRdh54 signaling function in adaption evident in the single mutant.
Biochemically, ScRdh54 is exceedingly similar to ScRad54, and it appears that their specificity is mediated by their differential affinities for Rad51, Dmc1, and possibly other proteins (). Like ScRad54, ScRdh54 is a dsDNA-specific ATPase, albeit with apparently lower specific activity (). ScRdh54 translocates on dsDNA, with lower velocity (84 bp/min) and processivity than ScRad54, inducing the topological changes identified to be associated with ScRad54 translocation [93
]. In summary, the genetic, biochemical, and cytological data suggest some functional overlap between ScRdh54 and ScRad54, particularly in meiosis, but there are clearly unique functions for both proteins, e.g.
of ScRad54 in DNA repair in somatic cells and ScRdh54 in adaptation from DNA damage checkpoint-mediated cell cycle arrest.
Unlike the yeast Rad54 paralog, ScRdh54, the mammalian RAD54 paralog, hRAD54B, has a clear function in DNA repair. In human cells hRad54B colocalizes with hRad51 and hRad54 in the nucleus [95
]. The availability of a human cell line (HCT116) knocked out for both copies of RAD54B
directly demonstrated the relevance of hRad54B to HR, as these cells show reduced gene targeting frequencies and genomic instability [96
]. Importantly, hRAD54B-deficiency is synthetically lethal with depletion of the FEN1 nuclease, which is critical for lagging strand replication [97
].mics the synthetic lethality of rad27
encodes yeast FEN1) with any HR mutant in budding yeast [98
]. The conclusions from the genetic studies in human cells are corroborated by results with RAD54B-deficient mouse ES cells, which display sensitivity to IR and interstrand crosslinking agents (mitomycin C) [64
]. Interestingly, RAD54 RAD54B
double mutant ES cells do not exhibit increased mitomycin C sensitivity, whereas the double mutant animal does, suggesting possible tissue-specific expression and function of both proteins [64
]. Likewise, the yeast rad54 rdh54
double mutant is completely deficient in meiosis, whereas the RAD54 RAD54B
double mutant mouse is fertile [44
]. The overall biochemical properties of hRAD54B are highly similar to hRAD54, although the ATPase activity appears to be surprisingly low (, see below) [64
While it is tempting to draw the analogy that both yeast and vertebrates (human) contain one Rad54 protein (ScRad54, hRAD54) and one principal Rad54 paralog (ScRdh54, hRAD54B), the genetic data in yeast and mouse strongly suggest that hRAD54B is not the equivalent of ScRdh54. The phenotypes of the respective single mutants are too different, and the genetic analysis in mice documents clear differences to yeast.