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.
Accurate DNA replication and segregation are critical for maintaining genome integrity and suppressing cancer. Metnase and EEPD1 are DNA damage response (DDR) proteins frequently dysregulated in cancer and implicated in cancer etiology and tumor response to genotoxic chemo- and radiotherapy. Here, we examine the DDR in human cell lines with CRISPR/Cas9 knockout of Metnase or EEPD1. The knockout cell lines exhibit slightly slower growth rates, significant hypersensitivity to replication stress, increased genome instability and distinct alterations in DDR signaling. Metnase and EEPD1 are structure-specific nucleases. EEPD1 is recruited to and cleaves stalled forks to initiate fork restart by homologous recombination. Here, we demonstrate that Metnase is also recruited to stalled forks where it appears to dimethylate histone H3 lysine 36 (H3K36me2), raising the possibility that H3K36me2 promotes DDR factor recruitment or limits nucleosome eviction to protect forks from nucleolytic attack. We show that stalled forks are cleaved normally in the absence of Metnase, an important and novel result because a prior study indicated that Metnase nuclease is important for timely fork restart. A double knockout was as sensitive to etoposide as either single knockout, suggesting a degree of epistasis between Metnase and EEPD1. We propose that EEPD1 initiates fork restart by cleaving stalled forks, and that Metnase may promote fork restart by processing homologous recombination intermediates and/or inducing H3K36me2 to recruit DDR factors. By accelerating fork restart, Metnase and EEPD1 reduce the chance that stalled replication forks will adopt toxic or genome-destabilizing structures, preventing genome instability and cancer. Metnase and EEPD1 are overexpressed in some cancers and thus may also promote resistance to genotoxic therapeutics.
Robust genetic systems for the hyperthermophilic have facilitated the overexpression of native genes, enabled the addition of sequences encoding secretion signals, epitope, and affinity tags to coding regions, and aided the introduction of sequences encoding new proteins in these fast-growing fermentative heterotrophs. However, tightly controlled and easily manipulated systems facilitating regulated gene expression are limited for these hosts. Here, we describe an alternative method for regulatory control reliant on a-encoded functional riboswitch in the model archaeon Despite the hyperthermophilic growth temperatures, the proposed structure of the riboswitch conforms to a fluoride-responsive riboswitch encoded in many bacteria and similarly functions to regulate a component-conserved fluoride export pathway. Deleting components of the fluoride export pathway generates strains with increased fluoride sensitivity. The mechanism underlying regulated expression suggested that the riboswitch-encoding sequences could be utilized as a tunable expression cassette. When appended to a reporter gene, the riboswitch-mediated control system provides fluoride-dependent tunable regulatory potential, offering an alternative system for regulating gene expression. Riboswitch-regulated expression is thus ubiquitous in extant life and can be exploited to generate regulated expression systems for hyperthermophiles. Gene expression is controlled by a myriad of interconnected mechanisms that interpret metabolic states and environmental cues to balance cell physiology. Transcription regulation in is known to employ both typical repressors-operators and transcription activators to regulate transcription initiation in addition to the regulation afforded by chromatin structure. It was perhaps surprising that the presumed ancient mechanism of riboswitch-mediated regulation is found in and , but seemingly absent in We demonstrate here that a fluoride-responsive riboswitch functions to regulate a detoxification pathway in the hyperthermophilic archaeon The results obtained define a universal role for riboswitch-mediated regulation, adumbrate the presence of several riboswitch-regulated genes in, demonstrate the utility of RNA-based regulation at high temperatures, and provide a novel riboswitch-regulated expression system to employ in hyperthermophiles.
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