No abstract

Interplay between DNA repair of the oxidatively modified base 8-oxo-7,8-dihydroguanine (OG) and transcriptional activation has been documented in mammalian genes. Previously, we synthesized OG into the VEGF potential G-quadruplex sequence (PQS) in the coding strand of a luciferase promoter to identify that base excision repair (BER) unmasked the G-quadruplex (G4) fold for gene activation. In the present work, OG was site-specifically synthesized into a luciferase reporter plasmid to follow the time-dependent expression in mammalian cells when OG in the VEGF PQS context was located in the coding vs template strands of the luciferase promoter. Removal of OG from the coding strand by OG glycosylase-1 (OGG1)-mediated BER upregulated transcription. When OG was in the template strand in the VEGF PQS context, transcription was downregulated by a BER-independent process. The time course changes in transcription show that repair in the template strand was more efficient than repair in the coding strand. Promoters were synthesized with an OG:A base pair that requires repair on both strands to yield a canonical G:C base pair. By monitoring the up/down luciferase expression, we followed the timing of repair of an OG:A base pair occurring on both strands in mammalian cells in which one lesion resides in a G-quadruplex loop and one in a potential i-motif. Depending on the strand in which OG resides, coding vs template, this modification is an up/downregulator of transcription that couples DNA repair with transcriptional regulation.

ABSTRACT

The diastereomeric spiroiminodihydantoin-2′-deoxyribonucleoside (dSp) lesions resulting from 2′-deoxyguanosine (dG) or 8-oxo-7,8-dihydro-2′-deoxyguanosine (dOG) oxidation have generated much attention due to their highly mutagenic nature. Their propeller-like shape leads these molecules to display mutational profiles in vivo that are stereochemically dependent. However, there exist conflicting absolute configuration assignments arising from electronic circular dichroism (ECD) and NOESY-NMR experiments; thus, providing definitive assignments of the 3D structure of these molecules is of great interest. In the present body of work, we present data inconsistent with the reported ECD assignments for the dSp diastereomers in the nucleoside context, in which the first eluting isomer from a Hypercarb HPLC column was assigned to be the S configuration and the second was assigned the R configuration. The following experiments were conducted: (1) Determination of the diastereomer ratio of dSp products upon one-electron oxidation of dG in chiral hybrid or propeller G-quadruplexes that expose the re or si face to solvent, respectively, (2) absolute configuration analysis using vibrational circular dichroism (VCD) spectroscopy, (3) reinterpretation of the ECD experimental spectra using time-dependent density functional theory (TDDFT) with the inclusion of 12 explicit H-bonding waters around the Sp free bases, and (4) reevaluation of calculated specific rotations for the Sp enantiomers using the hydration model in the TDDFT calculations. These new insights provide a fresh look at the absolute configuration assignments of the dSp diastereomers in which the first eluting from a Hypercarb-HPLC column is (-)-(R)-dSp and the second is (+)-(S)-dSp. These assignments now provide the basis for understanding the biological significance of the stereochemical dependence of enzymes that process this form of DNA damage.

The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34 ؉ lin ؊ thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7 ؊ . CD34 ؉ lin ؊ thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-

Strands of DNA with four or more contiguous runs of 2'-deoxycytidine (dC) nucleotides have the potential to adopt i-motif folds, generally under mildly acidic conditions. Analysis of dC homo-oligonucleotide strands ranging in length from 10 to 30 nucleotides by five different pH-dependent methods identified a pattern in strand length vs stability. Beginning with dC, which does not fold, the transition pH (pH) increased with chain length with the addition of up to four nucleotides, after which the stability dramatically decreased, and the trend repeated this cycle up to dC. The analysis found dC strands of length 15, 19, 23, and 27 nucleotides (i.e., 4n-1) to have pH values >7.2 and thermal stabilities >37 °C at pH 7.0. Model studies using thymidine nucleotides to lock in i-motif loop lengths support the conclusion that the most stable dC i-motifs possess one nucleotide in each of the three loops and a core built of an even number of base pairs. The pattern identified from the model studies occurs with a frequency of four nucleotides at lengths of 15, 19, 23, and 27 in accordance with the results obtained for the dC strands. This observation led us to interrogate the human genome for dC runs. Inspection of the human genome indicates that dC runs are enriched in critical regions of the genome (promoters, UTRs, and introns), while being depleted in coding and intergenic regions, and these findings may have biological implications. Lastly, the ability to tune i-motif stabilities by the length of the strand might be harnessed for stimulus-responsive applications in DNA scaffolds, sensors, nanotechnology, and other chemical applications.

The cellular response to oxidative stress includes transcriptional changes, particularly for genes involved in DNA repair. Recently, our laboratory demonstrated that oxidation of 2'-deoxyguanosine (G) to 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) in G-rich potential G-quadruplex sequences (PQSs) in gene promoters impacts the level of gene expression up or down depending on the position of the PQS in the promoter. In the present report, bioinformatic analysis found that the 390 human DNA repair genes in the genome ontology initiative harbor 2936 PQSs in their promoters and 5'-untranslated regions (5'-UTRs). The average density of PQSs in human DNA repair genes was found to be nearly 2-fold greater than the average density of PQSs in all coding and noncoding human genes (7.5 vs 4.3 per gene). The distribution of the PQSs in the DNA repair genes on the nontranscribed (coding) vs transcribed strands reflects that of PQSs in all human genes. Next, literature data were interrogated to select 30 PQSs to catalog their ability to adopt G-quadruplex (G4) folds in vitro using five different experimental tests. The G4 characterization experiments concluded that 26 of the 30 sequences could adopt G4 topologies in solution. Last, four PQSs were synthesized into the promoter of a luciferase plasmid and cotransfected with the G4-specific ligands pyridostatin, Phen-DC3, or BRACO-19 in human cells to determine whether the PQSs could adopt G4 folds. The cell studies identified changes in luciferase expression when the G4 ligands were present, and the magnitude of the expression changes dependent on the PQS and the coding vs template strand on which the sequence resided. Our studies demonstrate PQSs exist at a high density in human DNA repair gene promoters and a subset of the identified sequences may fold in vitro and in vivo.

The NEIL3 DNA repair gene is induced in cells or animal models experiencing oxidative or inflammatory stress along with oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in the genome. We hypothesize that a G-rich promoter element that is a potential G-quadruplex-forming sequence (PQS) in NEIL3 is a site for introduction of OG with epigenetic-like potential for gene regulation. Activation occurs when OG is formed in the NEIL3 PQS located near the transcription start site. Oxidative stress either introduced by TNFα or synthetically incorporated into precise locations focuses the base excision repair process to read and catalyze removal of OG via OG-glycosylase I (OGG1), yielding an abasic site (AP). Thermodynamic studies showed that AP destabilizes the duplex, enabling a structural transition of the sequence to a G-quadruplex (G4) fold that positions the AP in a loop facilitated by the NEIL3 PQS having five G runs in which the four unmodified runs adopt a stable G4. This presents AP to apurinic/apyrimidinic endonuclease 1 (APE1) that poorly cleaves the AP backbone in this context according to in vitro studies, allowing the protein to function as a trans activator of transcription. The proposal is supported by chemical studies in cellulo and in vitro. Activation of NEIL3 expression via the proposed mechanism allows cells to respond to mutagenic DNA damage removed by NEIL3 associated with oxidative or inflammatory stress. Lastly, inspection of many mammalian genomes identified conservation of the NEIL3 PQS, suggesting this sequence was favorably selected to function as a redox switch with OG as the epigenetic-like regulatory modification.