Functional characterization and identification of mouse Rad51d splice variants

Gruver, Aaron M.; Yard, Brian D.; McInnes, Campbell; Rajesh, Changanamkandath; Pittman, Douglas L.

doi:10.1186/1471-2199-10-27

Cited by 10 publications

(16 citation statements)

References 48 publications

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“…EGFP-RNF138 localization, as expected, was predominantly nuclear and overlapped with the DAPI nuclear stain (data not shown). EGFP-RAD51D displayed nuclear localization but was also distributed throughout the nuclear and cytoplasmic compartments, consistent with our previous findings [35]. …”

Section: Resultssupporting

confidence: 92%

“…Y2H analysis was also performed using the RAD51D amino-terminal (residues 4–77) and core domain (residues 77–329) expression constructs (Figure 2A) [34,35]. It was shown previously that the RAD51D amino-terminal domain, specifically the linker region (residues 50–77), mediates binding with XRCC2, while the core domain is necessary for interaction with RAD51C.…”

Section: Resultsmentioning

confidence: 99%

“…Association was not detected between RAD51D 4–77 and RNF138 suggesting that RNF138 binds the RAD51D core domain, similar to RAD51C. To further narrow the region of interaction with RAD51D, the previously identified RAD51D splice variants were tested with RNF138 [35,41]. Of the seven variants, only Rad51d-Δ7b that lacks the final five amino acids of exon 7 (residues 219–223) induced β-galactosidase activity (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…A total of 3.3 × 10 7 clones were assayed (cfu/ml of diploids × resuspension volume). Liquid β-galactosidase assays were performed using ortho-nitrophenyl-β-galactopyranoside (ONPG: Sigma) [35]. Yeast two-hybrid expression vectors pGADT7 and pGBKT7 (Clontech) were co-transformed into Y187 haploids using the EZ Transformation Kit (Zymo).…”

Section: Methodsmentioning

confidence: 99%

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RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity

et al. 2016

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The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity

et al. 2016

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“…In spite of their indispensible role during DNA repair, few binding partners of the RAD51 paralogs have been identified. Yeast two-hybrid analysis, co-immunoprecipitation, mutation analysis and domain mapping of the RAD51 protein family have revealed protein-protein interactions among these gene products, but these are insufficient to explain the mechanism of action of these proteins in HR repair [16,[27][28][29][30][31]. In this study, we have used three different RAD51-like proteins, RAD51D, RAD51C and XRCC2, each known to function as a complex and participate in HR repair, for co-precipitation followed by mass spectroscopic analysis of their interacting partners.…”

Section: Discussionmentioning

confidence: 99%

The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis

Rajesh¹,

Gruver²,

Basrur³

et al. 2009

Proteomics

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The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co-precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1-D SDS-PAGE of co-precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co-precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.

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BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage

et al. 2013

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Homologous recombination plays an important role in the high-fidelity repair of DNA double-strand breaks. A central player in this process, RAD51, polymerizes onto single-stranded DNA and searches for homology in a duplex donor DNA molecule, usually the sister chromatid. Homologous recombination is a highly regulated event in mammalian cells: some proteins have direct enzymatic functions, others mediate or overcome rate-limiting steps in the process, and still others signal cell cycle arrest to allow repair to occur. While the human BRCA2 protein has a clear role in delivering and loading RAD51 onto single-stranded DNA generated after resection of the DNA break, the mechanistic functions of the RAD51 paralogs remain unclear. In this study, we sought to determine the genetic interactions between BRCA2 and the RAD51 paralogs during DNA DSB repair. We utilized siRNA-mediated knockdown of these proteins in human cells to assess their impact on the DNA damage response. The results indicate that loss of BRCA2 alone imparts a more severe phenotype than the loss of any individual RAD51 paralog and that BRCA2 is epistatic to each of the four paralogs tested.

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Functional characterization and identification of mouse Rad51d splice variants

Cited by 10 publications

References 48 publications

RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity

RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity

The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis

BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage

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