Analysis of mouse Rad54 expression and its implications for homologous recombination

Essers, Jeroen; Hendriks, Rudi W.; Wesoły, Joanna; Beerens, Cecile; Smit, Bep; Hoeijmakers, Jan H.J.; Wyman, Claire; Dronkert, Mies L. G.; Kanaar, Roland

doi:10.1016/s1568-7864(02)00110-6

Cited by 72 publications

(55 citation statements)

References 62 publications

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“…35). These results are consistent with previous observations that MYC and RAD51 protein levels increase with an increase in S-phase fraction (35,36).…”

Section: Resultssupporting

confidence: 93%

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

et al. 2010

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MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G 0 -G 1 and S-G 2 -M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization. Cancer Res; 70(21); 8748-59. ©2010 AACR.

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“…35). These results are consistent with previous observations that MYC and RAD51 protein levels increase with an increase in S-phase fraction (35,36).…”

Section: Resultssupporting

confidence: 93%

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

et al. 2010

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“…Thus, the maximal stimulation seen at equimolar amounts of Rad54 and Rad51 proteins in certain assays represents the limiting level rather than an obligatory requirement to form such saturated complexes. In this regard, it remains intriguing that the intracellular ratio of Rad54 protein to Rad51 protein for both yeast and mouse cells is approximately equimolar (46,47), a physiological finding that is consistent with our biochemical observations. The observation that stimulation saturates at lower than equimolar in some in vitro experiments can result from any number of factors.…”

Section: Discussionsupporting

confidence: 89%

A Novel Function of Rad54 Protein

Mazin¹,

Alexeev²,

Kowalczykowski³

2003

Journal of Biological Chemistry

157

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Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with singlestranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.Homologous recombination plays an essential role in repair of DNA double-stranded breaks, a lethal type of DNA damage. The mechanisms of double-stranded break repair by recombination have been extensively investigated at the genetic and biochemical levels (1-6). In yeast, genetic analysis reveals a set of genes, called the RAD52 epistasis group, that are directly involved in double-stranded break repair (7). This group includes the RAD50, RAD58/MRE11, XRS2, RPA1, RAD51, RAD52, RAD54, RAD55, RAD57, and RAD59 genes. Among the proteins encoded by these genes, the Rad51 protein is the most evolutionarily conserved; its homologues are spread from bacteria to mammals (8). The Rad51 protein family members possess a unique property; they form helical nucleoprotein filaments with DNA (9 -11) and promote DNA pairing and strand transfer, a basic step of homologous recombination (11-16).More recently, it was demonstrated that Rad52 protein, Rad54 protein, and the Rad55/Rad57 heterodimer stimulate DNA pairing promoted by Rad51 protein. The mechanistic studies revealed that stimulation by Rad52 protein and the Rad55/Rad57 proteins is at the early (presynaptic) stage of DNA strand exchange. Rad52 protein enhances the ability of Rad51 protein to displace RPA from ssDNA 1 (17)(18)(19)(20), as do the Rad55/Rad57 proteins, but by a different mechanism (21).The mechanism by which Rad54 protein stimulates the DNA pairing activity of Rad51 protein is different (22-28). It was found that, in contrast to other proteins that stimulate the DNA pairing activity of Rad51 protein, Rad54 protein acts at a later (synaptic) stage of DNA strand exchange (24, 29). Rad54 protein belongs to the Snf2/Swi2 group of proteins, which are involved in ATP-dependent remodeling of protein complexes, in...

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“…In contrast, chronic hypoxic conditions (72 h Â 0.2% O 2 ) only decreased survival on average by 18%. HR protein expression and function can be cell cycle specific and biased by altered cell cycle distribution after hypoxic gassing (37,38). In our model, hypoxia (0.2% O 2 ) did not decrease cell proliferation or change the cell cycle distribution (Fig.…”

Section: Resultsmentioning

confidence: 58%

Chronic Hypoxia Decreases Synthesis of Homologous Recombination Proteins to Offset Chemoresistance and Radioresistance

et al. 2008

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Hypoxic and/or anoxic tumor cells can have increased rates of mutagenesis and altered DNA repair protein expression. Yet very little is known regarding the functional consequences of any hypoxia-induced changes in the expression of proteins involved in DNA double-strand break repair. We have developed a unique hypoxic model system using H1299 cells expressing an integrated direct repeat green fluorescent protein (DR-GFP) homologous recombination (HR) reporter system to study HR under prolonged chronic hypoxia (up to 72 h under 0.2% O 2 ) without bias from altered proliferation, cell cycle checkpoint activation, or severe cell toxicity. We observed decreased expression of HR proteins due to a novel mechanism involving decreased HR protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O 2 as measured by the DR-GFP reporter system. This decrease in functional HR resulted in increased sensitivity to the DNA cross-linking agents mitomycin C and cisplatin but not to the microtubule-interfering agent, paclitaxel. Chronically hypoxic H1299 cells that had decreased functional HR were relatively radiosensitive [oxygen enhancement ratio (OER), 1.37] when compared with acutely hypoxic or anoxic cells (OER, 1.96-2.61). Using CAPAN1 cells isogenic for BRCA2 and siRNA to RAD51, we confirmed that the hypoxia-induced radiosensitivity was due to decreased HR capacity. Persistent down-regulation of HR function by the tumor microenvironment could result in low-fidelity DNA repair and have significant implications for response to therapy and genetic instability in human cancers. [Cancer Res 2008;68(2):605-14]

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Analysis of mouse Rad54 expression and its implications for homologous recombination

Cited by 72 publications

References 62 publications

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

A Novel Function of Rad54 Protein

Chronic Hypoxia Decreases Synthesis of Homologous Recombination Proteins to Offset Chemoresistance and Radioresistance

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