An O-Hydroxylamine-Coupled Alkaline Gel Electrophoresis Assay Demonstrates an Accumulation of Real Single Strand Breaks and Intact AP sites in Base Excision Repair Deficient Cells

April M. Single strand breaks (SSBs) are among 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 artifactual SSBs arising from cleaved alkali-labile sites. Here we developed a specific SSB assay using alkaline gel electrophoresis (AGE) coupled with an O-hydroxylamine, O-(Tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes AP sites to prevent their incision during alkaline DNA denaturation. DNA from DT40 and isogenic polymerase β null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE assay. The detection of true SSB formation was observed in each cell line with significantly greater formation observed in the null cells. Furthermore, a modification of the assay demonstrated the accumulation of intact AP sites in genomic DNA from both cell lines. OTX use represents a facile approach for assessing SSBs, whose benefits may also be applied to other established SSB assays. have never survived this ordeal. Also, to my family who love me flaws and all. List of Figures Page Figure 1 – Examining pH effect on DNA alkaline denaturation……………………... Figure 2 – OTX optimization for the protection of AP sites………………………… Figure 3 – SSB detection in DT40 and Pol β null cells exposed continuously to 1 mM MMS for up to 4 hours A – Indirect measurement through NAD(P)H depletion….………………… B – Gel electrophoresis visualization………..…………………………….… C – Intact and fragmented DNA analysis…………………………….……… Figure 4 – AP site detection in DT40 and Pol β null cells exposed continuously to 1 mM MMS for up to 4 hours A – Slot-blot analysis…………………………………………...…………… B – Gel electrophoresis visualization of DT40 cells………………………… C – Gel electrophoresis visualization of Pol β null cells……………….…… D – Intact and fragmented DNA analysis of DT40 cells……………….…… E – Intact and fragmented DNA analysis of Pol β null cells………….……...