Fidelity of a Bacterial DNA Polymerase in Microgravity, a Model for Human Health in Space

Long-term space missions will expose crew members, their cells as well as their microbiomes to prolonged periods of microgravity and ionizing radiation, environmental stressors for which almost no earth-based organisms have evolved to survive. Despite the importance of maintaining genomic integrity, the impact of these stresses on DNA polymerase-mediated replication and repair has not been fully explored. DNA polymerase fidelity and replication rates were assayed under conditions of microgravity generated by parabolic flight and compared to earth-like gravity. Upon commencement of a parabolic arc, primed synthetic single-stranded DNA was used as a template for one of two enzymes (Klenow fragment exonuclease+/−; with and without proofreading exonuclease activity, respectively) and were quenched immediately following the 20 s microgravitational period. DNA polymerase error rates were determined with an algorithm developed to identify experimental mutations. In microgravity Klenow exonuclease+ showed a median 1.1-fold per-base decrease in polymerization fidelity for base substitutions when compared to earth-like gravity (p = 0.02), but in the absence of proofreading activity, a 2.4-fold decrease was observed (p = 1.98 × 10−11). Similarly, 1.1-fold and 1.5-fold increases in deletion frequencies in the presence or absence of exonuclease activity (p = 1.51 × 10−7 and p = 8.74 × 10−13), respectively, were observed in microgravity compared to controls. The development of this flexible semi-autonomous payload system coupled with genetic and bioinformatic approaches serves as a proof-of-concept for future space health research.

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome

To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of Nsp14 exonuclease activity and the co-factor Nsp10. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. IMPDH2, which catalyzes the rate-limiting step of guanidine biosynthesis, was identified as a key mediator of the effect. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in presence of an IMPDH2 inhibitor. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

Xrn1 is a deNADding Enzyme Modulating Mitochondrial NAD Levels

The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5′-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5′-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that still retains its 5′-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and in turn intramitochondrial NAD levels. Our findings uncover a functional role for mitochondrial NAD-capped RNAs as a reservoir to maintain overall NAD homeostasis. We propose NAD-capped RNAs function as a cistern for mitochondrial NAD with Xrn1 serving as a rheostat for NAD-capped RNAs.

The PHP domain of PolX from Staphylococcus aureus aids high fidelity DNA synthesis through the removal of misincorporated deoxyribo-, ribo- and oxidized nucleotides

The X family is one of the eight families of DNA polymerases (dPols) and members of this family are known to participate in the later stages of Base Excision Repair. Many prokaryotic members of this family possess a Polymerase and Histidinol Phosphatase (PHP) domain at their C-termini. The PHP domain has been shown to possess 3′–5′ exonuclease activity and may represent the proofreading function in these dPols. PolX from Staphylococcus aureus also possesses the PHP domain at the C-terminus, and we show that this domain has an intrinsic Mn2+ dependent 3′–5′ exonuclease capable of removing misincorporated dNMPs from the primer. The misincorporation of oxidized nucleotides such as 8oxodGTP and rNTPs are known to be pro-mutagenic and can lead to genomic instability. Here, we show that the PHP domain aids DNA replication by the removal of misincorporated oxidized nucleotides and rNMPs. Overall, our study shows that the proofreading activity of the PHP domain plays a critical role in maintaining genomic integrity and stability. The exonuclease activity of this enzyme can, therefore, be the target of therapeutic intervention to combat infection by methicillin-resistant-Staphylococcus-aureus.

APE1 distinguishes DNA substrates in exonucleolytic cleavage by induced space-filling

The exonuclease activity of Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for processing matched/mismatched terminus in various DNA repair pathways and for removing nucleoside analogs associated with drug resistance. To fill in the gap of structural basis for exonucleolytic cleavage, we determine the APE1-dsDNA complex structures displaying end-binding. As an exonuclease, APE1 does not show base preference but can distinguish dsDNAs with different structural features. Integration with assaying enzyme activity and binding affinity for a variety of substrates reveals for the first time that both endonucleolytic and exonucleolytic cleavage can be understood by an induced space-filling model. Binding dsDNA induces RM (Arg176 and Met269) bridge that defines a long and narrow product pocket for exquisite machinery of substrate selection. Our study paves the way to comprehend end-processing of dsDNA in the cell and the drug resistance relating to APE1.

Activation of palindromes on a degradable modular grafting probe enables ultrasensitive detection of microRNAs

This work describes a single-stranded degradable modular grafting probe for analyzing microRNA-21. In the system, the exonuclease activity of phi 29 polymerase restrains the Sybr Green I/ssDNA induced background. The...