Accumulation of true single strand breaks and AP sites in base excision repair deficient cells

Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells. However, the neutralization of methoxyamine is required prior to usage. Here we developed a convenient, specific SSB assay using alkaline gel electrophoresis (AGE) coupled with a neutral O-hydroxylamine, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes abasic sites (AP sites) to prevent their alkaline incision while still allowing for strong alkaline DNA denaturation. DNA from DT40 and isogenic polymerase β null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE (OTX-AGE) assay. Time-dependent increases in SSBs were detected in each cell line with more extensive SSB formation in the null cells. These findings were supported by an assay that indirectly detects SSBs through measuring NAD(P)H depletion. An ARP-slot blot assay demonstrated a significant time-dependent increase in AP sites in both cell lines by 1 mM MMS compared to control. Furthermore, the Pol β-null cells displayed greater AP site formation than the parental DT40 cells. OTX use represents a facile approach for assessing SSB formation, whose benefits can also be applied to other established SSB assays.