Editing of the pre-mRNA for the DNA repair enzyme NEIL1 causes a lysine to arginine change in the lesion recognition loop of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form removes thymine glycol from duplex DNA 30 times more slowly than the form encoded in the genome, whereas editing enhances repair of the guanidinohydantoin lesion by NEIL1. In addition, we show that the NEIL1 recoding site is a preferred editing site for the RNA editing adenosine deaminase ADAR1. The edited adenosine resides in an A-C mismatch in a hairpin stem formed by pairing of exon 6 to the immediate upstream intron 5 sequence. As expected for an ADAR1 site, editing at this position is increased in human cells treated with interferon α. These results suggest a unique regulatory mechanism for DNA repair and extend our understanding of the impact of RNA editing.ADAR | nucleic acids | base excision repair | oxidative stress | DNA damage

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.bacterial signaling | surface sensing | second messengers | cyclic dinucleotides | fluorescent biosensor

Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all *

Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.

Thymine glycol (Tg), one of the oxidized bases formed in DNA by reactive oxygen species, is repaired by the DNA glycosylases such as NEIL1, NTH1 and Endo III. In our recent studies, we showed that NEIL1’s catalytic efficiency and lesion specificity are regulated by an RNA editing adenosine deamination reaction. In this study, we synthesized oligodeoxynucleotides containing 2′-fluorothymidine glycol with either ribo or arabino configuration and investigated the binding of these modified DNAs with the unedited and edited forms of human NEIL1 along with E. coli Endo III. For the two forms of hNEIL1, binding affinities to FTg-containing DNA were similar indicating the editing effect is more subtle than to simply alter substrate affinity. While the NEIL1 binding to FTg-containing DNAs was largely insensitive to C5 and 2′ stereochemistry, a preference was observed for the FTg-G pair over the FTg-A pair. In addition, we found that optimal binding is observed with Endo III and duplex DNA with riboFTg (5S) paired with dG. The modified DNAs reported here will provide useful tools for further characterizing the interaction between DNA repair glycosylases and thymine glycol containing DNA.