Twelve complete genomes of three novel coronaviruses-bat coronavirus HKU4 (bat-CoV HKU4), bat-CoV HKU5 (putative group 2c), and bat-CoV HKU9 (putative group 2d)-were sequenced. Comparative genome analysis showed that the various open reading frames (ORFs) of the genomes of the three coronaviruses had significantly higher amino acid identities to those of other group 2 coronaviruses than group 1 and 3 coronaviruses. Phylogenetic trees constructed using chymotrypsin-like protease, RNA-dependent RNA polymerase, helicase, spike, and nucleocapsid all showed that the group 2a and 2b and putative group 2c and 2d coronaviruses are more closely related to each other than to group 1 and 3 coronaviruses. Unique genomic features distinguishing between these four subgroups, including the number of papain-like proteases, the presence or absence of hemagglutinin esterase, small ORFs between the membrane and nucleocapsid genes and ORFs (NS7a and NS7b), bulged stem-loop and pseudoknot structures downstream of the nucleocapsid gene, transcription regulatory sequence, and ribosomal recognition signal for the envelope gene, were also observed. This is the first time that NS7a and NS7b downstream of the nucleocapsid gene has been found in a group 2 coronavirus. The high Ka/Ks ratio of NS7a and NS7b in bat-CoV HKU9 implies that these two group 2d-specific genes are under high selective pressure and hence are rapidly evolving. The four subgroups of group 2 coronaviruses probably originated from a common ancestor. Further molecular epidemiological studies on coronaviruses in the bats of other countries, as well as in other animals, and complete genome sequencing will shed more light on coronavirus diversity and their evolutionary histories.
Topoisomerases are crucial to solve DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3β (Top3β) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein deficient in Fragile X syndrome and known to regulate translation of mRNAs important for neuronal function and autism. Notably, the FMRP-Top3β interaction is abolished by a disease-associated FMRP mutation, suggesting that Top3β may contribute to pathogenesis of mental disorders. Top3β binds multiple mRNAs encoded by genes with neuronal functions related to schizophrenia and autism. Expression of one such gene, ptk2/FAK, is reduced in neuromuscular junctions of Top3β mutant flies. Synapse formation is defective in Top3β mutant flies and mice, as observed in FMRP mutant animals. Our findings suggest that Top3β acts as an RNA topoisomerase and works with FMRP to promote expression of mRNAs critical for neurodevelopment and mental health.
Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends.
Apart from bat-SARS-CoV, we have identified a novel group 1 coronavirus, bat-CoV HKU2, in Rhinolophus sinicus (Chinese horseshoe bats). Since it has been suggested that the receptor-binding motif (RBM) of SARS-CoV may have been acquired from a group 1 coronavirus, we conducted a surveillance study and identified bat-SARS-CoV and bat-CoV HKU2 in 8.7% and 7.5% respectively of R. sinicus in Hong Kong and Guangdong. Complete genome sequencing of four strains of bat-CoV HKU2 revealed the smallest coronavirus genome (27164 nucleotides) and a unique spike protein evolutionarily distinct from the rest of the genome. This spike protein, sharing similar deletions with other group 2 coronaviruses in its C-terminus, also contained a 15-amino acid peptide homologous to a corresponding peptide within the RBM of spike protein of SARS-CoV, which was absent in other coronaviruses except bat-SARS-CoV. These suggest a common evolutionary origin in the spike protein of bat-CoV HKU2, bat-SARS-CoV, and SARS-CoV.
The ATM (ataxia telangiectasia mutated) protein kinase is activated under physiological and pathological conditions that induce DNA double-strand breaks (DSBs). Loss of ATM or failure of its activation in humans and mice lead to defective cellular responses to DSBs, such as cell cycle checkpoints, radiation sensitivity, immune dysfunction, infertility and cancer predisposition. A widely used biological marker to identify the active form of ATM is the autophosphorylation of ATM at a single, conserved serine residue (Ser 1981 in humans; Ser 1987 in mouse). Here we show that Atm-dependent responses are functional at the organismal and cellular level in mice that express a mutant form of Atm (mutation of Ser to Ala at position 1987) as their sole Atm species. Moreover, the mutant protein does not exhibit dominant-negative interfering activity when expressed physiologically or overexpressed in the context of Atm heterozygous mice. These results suggest an alternative mode for stimulation of Atm by DSBs in which Atm autophosphorylation at Ser 1987, like trans-phosphorylation of downstream substrates, is a consequence rather than a cause of Atm activation.
BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C-terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM-deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA-binding domain that resembles the aCTD domain of bacterial RNA polymerase a; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA-binding interface for the BLM complex to restart stalled replication forks.
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