The Worldwide Search for the New Mutations in the RNA-Directed RNA Polymerase Domain of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, responsible for the current pandemic outbreak. In total, 200 genomes of the SARS-CoV-2 strains from four host organisms have been analyzed. To investigate the presence of the new mutations in the RNA-directed RNA Polymerase (RdRp) of SARS-CoV-2, we analyzed sequences isolated from different hosts, with particular emphasis on human isolates. We performed a search for the new mutations of the RdRp proteins and study how those newly identified mutations could influence RdRp protein stability. Our results revealed 25 mutations in Rhinolophus sinicus, 1 in Mustela lutreola, 6 in Homo sapiens, and none in Mus musculus RdRp proteins of the SARS-CoV-2 isolates. We found that P323L is the most common stabilising radical mutation in human isolates. Also, we described several unique mutations, specific for studied hosts. Therefore, our data suggest that new and emerging variants of the SARS-CoV-2 RdRp have to be considered for the development of effective therapeutic agents and treatments.

The possible fidelity-speed-proofreading cost trade-offs in DNA replication due to the exonuclease proofreading

DNA replication is a high-fidelity information-copying processes which is realized by DNA polymerase (DNAP). The high fidelity was explained on the basis of the well-known kinetic-proofreading mechanism (KPR), under which the so-called fidelity-speed trade-off was studied theoretically. However, numerous biochemical experiments have shown that the high fidelity of DNA replication is achieved due to the initial discrimination of polymerase domain of DNAP, as well as the proofreading of the exonuclease domain of DNAP. This exonuclease-proofreading mechanism (EPR) is totally different from KPR. So the trade-off issues are worth being re-examined under EPR. In this paper, we use the first-passage method recently proposed by us to discuss the possible trade-offs in DNA replication under EPR. We show that there could be no fidelity-speed trade-off under EPR, i.e., the fidelity and the speed can be simultaneously enhanced by EPR in a large range of kinetic parameters. This provides a new perspective to understand the experimental data of the exonuclease activity of T7 DNAP and T4 DNAP. We also show that there exists the fidelity-proofreading cost trade-off, i.e., the fidelity is enhanced at the cost of increasing the futile hydrolysis of dNTP. A possible way to avoid this trade-off is to regulate the rate of DNAP translocation: slowing down the forward translocation (in the presence of the terminal mismatch) can enhance the fidelity without changing the speed and the proofreading cost. Our theoretical analysis offers deeper insights on the kinetics-function relation of DNAP.PACS numbers: 82.39.-k, 87.15.Rn, 87.16.A-

Identification of Uncharacterized Components of Prokaryotic Immune Systems and Their Diverse Eukaryotic Reformulations

ABSTRACT Nucleotide-activated effector deployment, prototyped by interferon-dependent immunity, is a common mechanistic theme shared by immune systems of several animals and prokaryotes. Prokaryotic versions include CRISPR-Cas with the CRISPR polymerase domain, their minimal variants, and systems with second messenger oligonucleotide or dinucleotide synthetase (SMODS). Cyclic or linear oligonucleotide signals in these systems help set a threshold for the activation of potentially deleterious downstream effectors in response to invader detection. We establish such a regulatory mechanism to be a more general principle of immune systems, which can also operate independently of such messengers. Using sensitive sequence analysis and comparative genomics, we identify 12 new prokaryotic immune systems, which we unify by this principle of threshold-dependent effector activation. These display regulatory mechanisms paralleling physiological signaling based on 3′-5′ cyclic mononucleotides, NAD+-derived messengers, two- and one-component signaling that includes histidine kinase-based signaling, and proteolytic activation. Furthermore, these systems allowed the identification of multiple new sensory signal sensory components, such as a tetratricopeptide repeat (TPR) scaffold predicted to recognize NAD+-derived signals, unreported versions of the STING domain, prokaryotic YEATS domains, and a predicted nucleotide sensor related to receiver domains. We also identify previously unrecognized invader detection components and effector components, such as prokaryotic versions of the Wnt domain. Finally, we show that there have been multiple acquisitions of unidentified STING domains in eukaryotes, while the TPR scaffold was incorporated into the animal immunity/apoptosis signal-regulating kinase (ASK) signalosome. IMPORTANCE Both prokaryotic and eukaryotic immune systems face the dangers of premature activation of effectors and degradation of self-molecules in the absence of an invader. To mitigate this, they have evolved threshold-setting regulatory mechanisms for the triggering of effectors only upon the detection of a sufficiently strong invader signal. This work defines general templates for such regulation in effector-based immune systems. Using this, we identify several previously uncharacterized prokaryotic immune mechanisms that accomplish the regulation of downstream effector deployment by using nucleotide, NAD+-derived, two-component, and one-component signals paralleling physiological homeostasis. This study has also helped identify several previously unknown sensor and effector modules in these systems. Our findings also augment the growing evidence for the emergence of key animal immunity and chromatin regulatory components from prokaryotic progenitors.

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5′ exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3′-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3′ exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

MITOCHONDRIA AS REVERSIBLE REGULATORS OF AGING ASSOCIATED SKIN WRINKLES AND HAIR LOSS IN MICE

To evaluate the consequences of the decline in mtDNA content associated with aging we have created an inducible mouse model expressing, in the polymerase domain of POLG1, a dominant-negative mutation that induces depletion of mtDNA. We utilized this inducible mouse model to modulate mitochondrial function by depleting and repleting the mtDNA content. We demonstrate that, in mice, ubiquitous expression of dominant-negative mutant POLG1 leads to 1) reduction of mtDNA content in skin, 2) skin wrinkles, and 3) hair loss. By turning off the mutant POLG1 transgene expression in the whole animal, the skin and hair phenotypes revert to normal after repletion of mtDNA. Thus, we have developed whole-animal mtDNA depleter-repleter mice. These mice present evidence that mtDNA homeostasis is involved in skin aging phenotype and loss of hair and provide an unprecedented opportunity to create tissue-specific mitochondrial modulation to determine the role of the mitochondria in a particular tissue.

Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

Transcriptomic Analysis of Mucor irregularis Containing a Negative Single-Stranded RNA Mycovirus

The fungus Mucor irregularis is a causative agent of mucormycosis. The transcriptome analysis of the isolated M. irregularis strain C3B revealed the presence of an RNA polymerase domain of a negative-polarity RNA virus. In this work, we describe the gene ontology-based annotation of the Mucor irregularis transcriptome, which includes a putative RNA mycovirus.