Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1
Name:
Article-Cell_Repor-Sequential-2013-06-06.pdf
Size:
3.205Mb
Format:
PDF
Description:
Article - Full Text
Type
Article
KAUST Department
Biological and Environmental Sciences and Engineering (BESE) DivisionBioscience Program
Enrichment Program
Laboratory of DNA Replication and Recombination
Physical Science and Engineering (PSE) Division
Date
2013-06-06Online Publication Date
2013-06-06Print Publication Date
2013-06Permanent link to this record
http://hdl.handle.net/10754/334510Metadata
Show full item recordAbstract
Human flap endonuclease 1 (FEN1), one of the structure-specific 5' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5' nuclease mechanisms. This is achieved by coordinating threading of the 5' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.Citation
Sobhy MA, Joudeh LI, Huang X, Takahashi M, Hamdan SM (2013) Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1. Cell Reports 3: 1785-1794. doi:10.1016/j.celrep.2013.05.001.Publisher
Elsevier BVJournal
Cell ReportsPubMed ID
23746444Scopus Count
The following license files are associated with this item:
Except where otherwise noted, this item's license is described as http://creativecommons.org/licenses/by/3.0/
Related items
Showing items related by title, author, creator and subject.
-
Antioxidant enzymes expression in lymphocytes of patients undergoing carotid endarterectomy(Medical Hypotheses, Elsevier BV, 2019-10-03) [Article]To remedy carotid artery stenosis and prevent stroke surgical intervention is commonly used, and the gold standard being carotid endarterectomy (CEA). During CEA cerebrovascular hemoglobin oxygen saturation decreases and when this decrease reaches critical levels it leads to cerebral hypoxia that causes neuronal damage. One of the proposed mechanism that affects changes during CEA and contribute to acute brain ischemia (ABI) is oxidative stress. The increased production of reactive oxygen species and reactive nitrogen species during ABI may cause an unregulated inflammatory response and further lead to structural and functional injury of neurons. Antioxidant activity are involved in the protection against neuronal damage after cerebral ischemia. We hypothesized that neuronal injury and poor outcomes in patients undergoing CEA may be results of oxidative stress that disturbed function of antioxidant enzymes and contributed to the DNA damage in lymphocytes.
-
Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes(ACS Omega, American Chemical Society (ACS), 2021-03-15) [Article]One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.
-
A RALDH-like enzyme involved in Fusarium verticillioides development(Fungal Genetics and Biology, Elsevier BV, 2015-12-16) [Article]Retinaldehyde dehydrogenases (RALDHs) convert retinal to retinoic acid, an important chordate morphogen. Retinal also occurs in some fungi, such as Fusarium and Ustilago spp., evidenced by the presence of rhodopsins and β–carotene cleaving, retinal-forming dioxygenases. Based on the assumption that retinoic acid may also be formed in fungi, we searched the Fusarium protein databases for RALDHs homologs, focusing on Fusarium verticillioides. Using crude lysates of Escherichia coli cells expressing the corresponding cDNAs, we checked the capability of best matches to convert retinal into retinoic acid in vitro. Thereby, we identified an aldehyde dehydrogenase, termed CarY, as a retinoic acid-forming enzyme, an activity that was also exerted by purified CarY. Targeted mutation of the carY gene in F. verticillioides resulted in alterations of mycelia development and conidia morphology in agar cultures, and reduced capacity to produce perithecia as a female in sexual crosses. Complementation of the mutant with a wild-type carY allele demonstrated that these alterations are caused by the lack of CarY. However, retinoic acid could not be detected by LC-MS analysis either in the wild type or the complemented carY strain in vivo, making elusive the connection between CarY enzymatic activity and retinoic acid formation in the fungus.
