Structural basis for substrate discrimination by E. coli repair enzyme, AlkB.

E. coli AlkB, a repair enzyme of the dioxygenase family, catalyses the removal of mutagenic methylated nucleotides from the genome. Known for substrate promiscuity, AlkB's catalytic mechanism and conformational changes accompanying substrate binding have been extensively dissected. However, the structural parameters of various substrates governing their recognition by AlkB still remain elusive. In this work, through solution-state vibrational spectra of methylated substrates bound to AlkB in combination with computational analysis, we show that the recognition specificity is dictated by the protonation states of the substrates. Specificity is conferred predominantly through hydrogen bonding and cation-π interactions. Furthermore, we report on the interaction of AlkB with normal, unmodified nucleotides, wherein the presence of an exocyclic amino group serves as an essential criterion for the initial process of substrate recognition. Taken together, these results provide a rationale for structural determinants of substrate specificity as well as mode of lesion discrimination employed by AlkB.

Mechanism of Discrimination of 8-Oxoguanosine versus Guanosine by Escherichia coli Fpg.

The mutagenic 8-oxoguanosine monophosphate, the predominant product of DNA oxidation, is excised by formamidopyrimidine glycosylase (Fpg) in bacteria. The mechanism of recognition of 8-oxodG, which differs subtly from its normal counterpart, guanosine monophosphate (dG), by Escherichia coli Fpg remains elusive due to the lack of structural data of E. coli Fpg bound to 8-oxodG. Here, we present solution-state structure of 8-oxodG oligomer bound to E. coli E3Q Fpg using UV resonance Raman (UVRR) spectroscopy. The vibrational spectra report on the π-stacking and hydrogen bonding interactions established by 8-oxodG with E. coli E3Q Fpg. Furthermore, we report on the interactions of E. coli E3Q Fpg with the normal, undamaged nucleotide, dG. We show that E. coli Fpg recognizes 8-oxodG and dG through their C2-amino group but only 8-oxodG forms extensive contacts with E. coli Fpg. Our findings provide a basis for mechanism of lesion recognition by E. coli Fpg.

Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing.

Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging.

RNA Polymerase II cluster dynamics predict mRNA output in living cells.

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous ß-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.

Methylation stabilizes the imino tautomer of dAMP and amino tautomer of dCMP in solution.

Alkylating agents cause methylation of adenosine and cytidine in DNA to generate 1-methyladenosine and 3-methylcytidine. These modified nucleosides can serve as regulators of cells or can act as agents of mutagenesis depending on the context and the partner enzymes. Solution structures and the chemical interactions with enzymes that lead to their recognition are of inherent interest. At physiological pH, 1-methyladenosine and 3-methylcytidine are presumed to be in the protonated amino forms in the literature. We report the structures, ionization states, and UV resonance Raman spectra of both substrates over a range of pH (2.5-11.0). The Raman excitation wavelength was tuned to selectively enhance Raman scattering from the nucleobase (260 nm) and further specifically from the imino form (210 nm) of 1-me-dAMP. We find that contrary to the general assumption, 1-me-dAMP is present in its neutral imino form at physiological pH and 3-me-dCMP is in the amino form.

Solution structure of the DNA damage lesion 8-oxoguanosine from ultraviolet resonance Raman spectroscopy.

UV radiation and reactive byproducts of cellular metabolism are constant threats to genomic stability. A frequent consequence is the oxidation of DNA nucleobases, especially guanine to 8-oxoguanosine. This highly mutagenic lesion can form base pairs with other nucleobases, does not significantly distort the DNA structure, and remains unnoticed by DNA polymerases. Detection and biophysical studies of modified nucleobases is challenging because they are not fluorescent and have broad electronic spectra that overlap with those of normal bases. The structure of 8-oxoguanosine and its anion in solution has been contentious in the literature. Using ultraviolet excitation in resonance with the nucleobase, we have obtained the Raman spectra of 8-oxoguanosine. The stable tautomer in solution is unequivocally identified as the diketone form. We show that, at high pH, 8-oxoguanosine gets deprotonated to form an anion through loss of the N1 proton from the pyrimidine ring. The enol form is never populated to a detectable level. Raman spectra are supported by density functional theoretical calculations and a complete normal-mode analysis to identify bands that can be used as reporters of protein-nucleobase interactions. We have demonstrated that UVRR spectra provide unprecedented information on the solution-state structures of modified nucleobases.