Mechanistic Insights into RAD51-associated Protein 1 (RAD51AP1) Action in Homologous DNA Repair

Dunlop, Myun Hwa; Dray, Eloïse; Zhao, Weixing; Filippo, Joseph San; Tsai, Miaw Sheue; Leung, Stanley G.; Schild, David; Wiese, Claudia; Sung, Patrick

doi:10.1074/jbc.c112.352161

Cited by 40 publications

(83 citation statements)

References 14 publications

(31 reference statements)

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“…Interaction-defective mutants of RAD51AP1, including a C-terminal truncation that still binds efficiently to D-loops, fail to stimulate D-loop formation. RAD51AP1 contains both N-and Cterminal DNA binding domains that contribute to its function in Dloop formation [1232]. RAD51AP1-depleted cells have normal RAD51 focus formation [1230,1231], which is consistent with RAD51AP1 acting at the stage of D-loop formation after RAD51 presynaptic filament formation.…”

Section: Rad51ap1mentioning

confidence: 76%

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

Thompson

2012

Mutation Research/Reviews in Mutation Research

317

276

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Section: Rad51ap1mentioning

confidence: 76%

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

Thompson

2012

Mutation Research/Reviews in Mutation Research

317

276

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“…Forward transfections with siRNA were carried out using Lipofectamine RNAiMAX reagent (Invitrogen), essentially as recommended by the manufacturer. Down-regulation of RAD51AP1 was mediated by RAD51AP1-directed siRNAs with the target sense sequences 5′-CCTCATATCTCTAATTGCA-3′ (here: AP1_1) and 5′-TGAACAATCTCCGGAAAGA-3′ (here: AP1_2) and as previously described [3–5]. For negative control siRNA, the following target sequence was used: 5′-GATTCGAACGTGTCACGTC-3′, as previously described [6].…”

Section: Methodsmentioning

confidence: 99%

RAD51AP1 -deficiency in vertebrate cells impairs DNA replication

et al. 2014

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RAD51-associated protein 1 (RAD51AP1) is critical for homologous recombination (HR) by interacting with and stimulating the activities of the RAD51 and DMC1 recombinases. In human somatic cells, knockdown of RAD51AP1 results in increased sensitivity to DNA damaging agents and to impaired HR, but the formation of DNA damage-induced RAD51 foci is unaffected. Here, we generated a genetic model system, based on chicken DT40 cells, to assess the phenotype of fully inactivated RAD51AP1 in vertebrate cells. Targeted inactivation of both RAD51AP1 alleles has no effect on either viability or doubling-time in undamaged cells, but leads to increased levels of cytotoxicity after exposure to cisplatin or to ionizing radiation. Interestingly, ectopic expression of GgRAD51AP1, but not of HsRAD51AP1 is able to fully complement in cell survival assays. Notably, in RAD51AP1-deficient DT40 cells the resolution of DNA damage-induced RAD51 foci is greatly slowed down, while their formation is not impaired. We also identify, for the first time, an important role for RAD51AP1 in counteracting both spontaneous and DNA damage-induced replication stress. In human and in chicken cells, RAD51AP1 is required to maintain wild type speed of replication fork progression, and both RAD51AP1-depleted human cells and RAD51AP1-deficient DT40 cells respond to replication stress by a slow-down of replication fork elongation rates. However, increased firing of replication origins occurs in RAD51AP1−/− DT40 cells, likely to ensure the timely duplication of the entire genome. Taken together, our results may explain why RAD51AP1 commonly is overexpressed in tumor cells and tissues, and we speculate that the disruption of RAD51AP1 function could be a promising approach in targeted tumor therapy.

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“…4,5,[19][20][21] Unlike the RAD51 paralogs (e.g. RAD51C), depletion of RAD51AP1 does not affect the foci formation of RAD51.…”

Section: Discussionmentioning

confidence: 99%

“…Knockdown of RAD51AP1 in human cells or knockout in DT40 cells leads to enhanced sensitivity to IR or DNA interstrand crosslinking agents. 4,5,18 RAD51AP1 is not essential for foci formation of RAD51, 4,5,18 but by directly associating with RAD51 and DNA, it stimulates the RAD51-mediated D-loop formation, 4,5,19 which is an intermediate structure during HR repair. Thus, RAD51AP1 is suggested to act downstream of the RAD51-ssDNA nucleoprotein filament formation.…”

Section: Introductionmentioning

confidence: 99%

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

et al. 2016

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USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interactiondeficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

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Mechanistic Insights into RAD51-associated Protein 1 (RAD51AP1) Action in Homologous DNA Repair

Cited by 40 publications

References 14 publications

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography

RAD51AP1 -deficiency in vertebrate cells impairs DNA replication

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

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