RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii

Journal Article · Mon Jul 17 00:00:00 EDT 2023 · mBio (Online)

DOI:https://doi.org/10.1128/mbio.00852-23· OSTI ID:1989957

Jia, Huiyong ^[1]; Dantuluri, Swathi ^[1]; Margulies, Shae ^[1]; Smith, Victoria ^[2]; Lever, Rebecca ^[2]; Allers, Thorsten ^[2]; Koh, Jin ^[3]; Chen, Sixue ^[4]; Maupin-Furlow, Julie A.  ^[5]; Gribaldo, ed., Simonetta; Clouet-d'Orval, ed., Beatrice

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1. Department of Microbiology and Cell Science, Institute of Food and Agricultural Science, University of Florida , Gainesville, Florida, USA
2. School of Life Sciences, University of Nottingham , Nottingham, United Kingdom
3. Proteomics and Mass Spectrometry, Interdisciplinary Center for Biotechnology Research, University of Florida , Gainesville, Florida, USA
4. Proteomics and Mass Spectrometry, Interdisciplinary Center for Biotechnology Research, University of Florida , Gainesville, Florida, USA, Genetics Institute, University of Florida , Gainesville, Florida, USA, Department of Biology, College of Liberal Arts and Sciences, University of Florida , Gainesville, Florida, USA
5. Department of Microbiology and Cell Science, Institute of Food and Agricultural Science, University of Florida , Gainesville, Florida, USA, Genetics Institute, University of Florida , Gainesville, Florida, USA

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii , an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3′−5′ exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5′−3′ exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identifiedidentifiedidentifiedin the archaeon Haloferax volcanii , which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3′−5′ exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5′−3′ exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

• Sponsoring Organization: USDOE
• Grant/Contract Number: FG02-05ER15650
• OSTI ID: 1989957
• Journal Information: mBio (Online), Journal Name: mBio (Online); ISSN 2150-7511
• Publisher: American Society for Microbiology
• Country of Publication: United States
• Language: English