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-