Abstract
Bacterial nuclease RecJ, which exists in almost all bacterial species, specifically degrades single-stranded (ss) DNA in the 5′ to 3′ direction. Some archaeal phyla, except Crenarchaea, also encode RecJ homologs. Compared with bacterial RecJ, archaeal RecJ exhibits a largely different amino acid sequence and domain organization. Archaeal RecJs from Thermococcus kodakarensis and Pyrococcus furiosus show 5′→3′ exonuclease activity on ssDNA. Interestingly, more than one RecJ exists in some Euryarchaeota classes, such as Methanomicrobia, Methanococci, Methanomicrobia, Methanobacteria, and Archaeoglobi. Here we report the biochemical characterization of two RecJs from Methanocaldococcus jannaschii, the long RecJ1 (MJ0977) and short RecJ2 (MJ0831) to understand their enzymatic properties. RecJ1 is a 5′→3′ exonuclease with a preference to ssDNA; however, RecJ2 is a 3′→5′ exonuclease with a preference to ssRNA. The 5′ terminal phosphate promotes RecJ1 activity, but the 3′ terminal phosphate inhibits RecJ2 nuclease. Go-Ichi-Ni-San (GINS) complex does not interact with two RecJs and does not promote their nuclease activities. Finally, we discuss the diversity, function, and molecular evolution of RecJ in archaeal taxonomy. Our analyses provide insight into the function and evolution of conserved archaeal RecJ/eukaryotic Cdc45 protein.
Highlights
Nucleases, including endonuclease and exonuclease, play important roles in DNA recombination and repair, degradation and recycling of DNA and RNA, and maturation of RNA and Okazaki fragments [1]
Genes encoding for the archaeal RecJ nucleases (MJ0831 and MJ0977) and GINS (MJ0248) were amplified from M. jannaschii genomic DNA by polymerase chain reaction (PCR) using their respective primers (Table S1) and inserted into pDEST17 or pET28-sumo, as described previously [17]
The two MjaRecJs have seven conserved motifs (I–VII), such as DHH and DHHA1, which are common among many DHH phosphodiesterase families
Summary
Nucleases, including endonuclease and exonuclease, play important roles in DNA recombination and repair, degradation and recycling of DNA and RNA, and maturation of RNA and Okazaki fragments [1]. The family 1 DHH phosphodiesterase includes three subfamilies: bacterial RecJ, archaeal RecJ and eukaryotic Cdc protein. Bacterial RecJ nuclease shows both single-stranded DNA (ssDNA)-specific 50 →30 exonuclease and deoxyribose phosphatase (dRPase) activities [3]. An essential replication initiation protein whose site-mutations result in partial defect in DNA replication [21], shows low-sequence similarity to the conserved catalytic core of the RecJ nuclease subfamily; Cdc lacks most of the conserved motifs and residues that are essential for prokaryotic enzymatic activity [4,22]. To fully understand the enzymatic properties of two M. jannaschii RecJs, we recombinantly expressed, purified and biochemically characterized them in detail Both RecJs are single-stranded DNA/RNA specific nucleases. Our results provide new clues to understand the functions of archaeal RecJ in nucleic acid metabolism and its evolution relationship with bacterial RecJ and eukaryotic Cdc protein
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