RecQ helicases play an important role in the maintenance of genomic stability in pro- and eukaryotes. This is highlighted by the human genetic diseases Werner, Bloom's and Rothmund-Thomson syndrome, caused by respective mutations in three of the five human RECQ genes. The highest numbers of RECQ homologous genes are found in plants, e.g. seven in Arabidopsis thaliana. However, only limited information is available on the functions of plant RecQ helicases, and no biochemical characterization has been performed. Here, we demonstrate that AtRECQ2 is a (d)NTP-dependent 3'-->5' DNA helicase. We further characterized its basal properties and its action on various partial DNA duplexes. Importantly, we demonstrate that AtRECQ2 is able to disrupt recombinogenic structures: by disrupting various D-loop structures, AtRECQ2 may prevent non-productive recombination events on the one hand, and may channel repair processes into non-recombinogenic pathways on the other hand, thus facilitating genomic stability. We show that a synthetic partially mobile Holliday junction is processed towards splayed-arm products, possibly indicating a branch migration function for AtRECQ2. The biochemical properties defined in this work support the hypothesis that AtRECQ2 might be functionally orthologous to the helicase part of the human RecQ homologue HsWRN.
Cavally virus (CavV) and related viruses in the family Mesoniviridae diverged profoundly from other nidovirus lineages but largely retained the characteristic set of replicative enzymes conserved in the Coronaviridae and Roniviridae. The expression of these enzymes in virus-infected cells requires the extensive proteolytic processing of two large replicase polyproteins, pp1a and pp1ab, by the viral 3C-like protease (3CL pro ). Here, we show that CavV 3CL pro autoproteolytic cleavage occurs at two N-terminal (N1 and N2) and one C-terminal (C1) processing site(s). The mature form of 3CL pro was revealed to be a 314-residue protein produced by cleavage at FKNK 1386 ԽSAAS (N2) and YYNQ 1700 ԽSATI (C1). Site-directed mutagenesis data suggest that the mesonivirus 3CL pro employs a catalytic Cys-His dyad comprised of CavV pp1a/pp1ab residues Cys-1539 and His-1434. The study further suggests that mesonivirus 3CL pro substrate specificities differ from those of related nidovirus proteases. The presence of Gln (or Glu) at the P1 position was not required for cleavage, although residues that control Gln/Glu specificity in related viral proteases are retained in the CavV 3CL pro sequence. Asn at the P2 position was identified as a key determinant for mesonivirus 3CL pro substrate specificity. Other positions, including P4 and P1=, each are occupied by structurally related amino acids, indicating a supportive role in substrate binding. Together, the data identify a new subgroup of nidovirus main proteases and support previous conclusions on phylogenetic relationships between the main nidovirus lineages. IMPORTANCEMesoniviruses have been suggested to provide an evolutionary link between nidovirus lineages with small (13 to 16 kb) and large (26 to 32 kb) RNA genome sizes, and it has been proposed that a specific set of enzymes, including a proofreading exoribonuclease and other replicase gene-encoded proteins, play a key role in the major genome expansion leading to the currently known lineages of large nidoviruses. Despite their smaller genome size (20 kb), mesoniviruses retained most of the replicative domains conserved in large nidoviruses; thus, they are considered interesting models for studying possible key events in the evolution of RNA genomes of exceptional size and complexity. Our study provides the first characterization of a mesonivirus replicase geneencoded nonstructural protein. The data confirm and extend previous phylogenetic studies of mesoniviruses and related viruses and pave the way for studies into the formation of the mesonivirus replication complex and functional and structural studies of its functional subunits.
Members of the conserved RecQ helicase family are important for the preservation of genomic stability. Multiple RecQ homologs within one organism raise the question of functional specialization. Whereas five different homologs are present in humans, the model plant Arabidopsis (Arabidopsis thaliana) carries seven RecQ homologs in its genome. We performed biochemical analysis of AtRECQ3, expanded upon a previous analysis of AtRECQ2, and compared their properties. Both proteins differ in their domain composition. Our analysis demonstrates that they are 3# to 5# helicases with similar activities on partial duplex DNA. However, they promote different outcomes with synthetic DNA structures that mimic Holliday junctions or a replication fork. AtRECQ2 catalyzes Holliday junction branch migration and replication fork regression, while AtRECQ3 cannot act on intact Holliday junctions. The observed reaction of AtRECQ3 on the replication fork is in line with unwinding the lagging strand. On nicked Holliday junctions, which have not been intensively studied with RecQ helicases before, AtRECQ3, but not AtRECQ2, shows a clear preference for one unwinding mechanism. In addition, AtRECQ3 is much more efficient at catalyzing DNA strand annealing. Thus, AtRECQ2 and AtRECQ3 are likely to perform different tasks in the cell, and AtRECQ3 differs in its biochemical properties from all other eukaryotic RECQ helicases characterized so far.RecQ helicases are important players in the maintenance of genomic stability in prokaryotes and eukaryotes (for reviews covering several aspects of RecQ helicases, see
Genetic and biochemical analyses of SRS2 homologs in fungi indicate a function in the processing of homologous recombination (HR) intermediates. To date, no SRS2 homologs have been described and analyzed in higher eukaryotes. Here, we report the first biochemical characterization of an SRS2 homolog from a multicellular eukaryote, the plant Arabidopsis thaliana. We studied the basic properties of AtSRS2 and were able to show that it is a functional 3′- to 5′-helicase. Furthermore, we characterized its biochemical function on recombinogenic intermediates and were able to show the unwinding of nicked Holliday junctions (HJs) and partial HJs (PX junctions). For the first time, we demonstrated strand annealing activity for an SRS2 homolog and characterized its strand pairing activity in detail. Our results indicate that AtSRS2 has properties that enable it to be involved in different steps during the processing of recombination intermediates. On the one hand, it could be involved in the unwinding of an elongating invading strand from a donor strand, while on the other hand, it could be involved in the annealing of the elongated strand at a later step.
Mesoniviridae are a family of insect RNA viruses that diverged profoundly from other families of the Nidovirales. Mesonivirus replicative proteins are produced from large polyprotein (pp) precursors (pp1a and pp1ab) through proteolytic cleavage by the viral 3C-like protease (3CL pro ) and, possibly, other proteases. Using recombinant forms of the Cavally virus 3CL pro and pp1a/pp1ab-derived substrates, we characterized 3CL pro cleavage sites in mesonivirus polyproteins. Our data lead us to suggest that 3CL pro cleaves the central and C-proximal regions of mesonivirus pp1a/pp1ab at 12 conserved sites. Compared to other nidovirus homologues, the mesonivirus 3CL pro features a distinct substrate specificity, with asparagine at P2 being a major specificity determinant. Furthermore, we provide evidence that expression of the ORF1b-encoded part of pp1ab involves a À1 ribosomal frameshift at a conserved GGAUUUU heptanucleotide sequence in the ORF1a/1b overlap region. Taken together, the study identifies critical steps in the expression and maturation of mesonivirus replicative proteins.
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