A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

Abstract

A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.

Interaction of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage, DNA Cleavage Inhibition, and Formation of Ligand–DNA Adducts

“…An alternative promising approach to block the BER pathway relies on DNA‐binding drugs that selectively bind to AP sites and thereby mask them, or render them unreactive with respect to APE1. Thus, methoxyamine (MX, or TRC102) and related alkoxyamines, which form covalent oxime adducts with AP sites, demonstrate synergy with DNA‐alkylating cytotoxic drugs, and a combination of MX with temozolomide is currently being evaluated for glioblastoma treatment in a Phase II clinical trial…”

“…[11][12][13] An alternative promising approach to block the BER pathway relies on DNA-binding drugs that selectively bind to AP sites and thereby mask them,o rr ender them unreactive with respect to APE1. Thus, methoxyamine (MX, or TRC102) and relatedalkoxyamines,w hich form covalent oxime adducts with AP sites, demonstrate synergyw ith DNA-alkylating cytotoxic drugs, [14,15] and ac ombination of MX with temozolomidei sc urrently being evaluated for glioblastoma treatment in aP hase II clinical trial. [16] Beyond covalent trapping of AP sites with reactive drugs, such as MX or analogues,s everal classes of noncovalentl igands were shown to selectively bind to AP sites in DNA and potentially hinder their recognition and processing by APE1.…”

A Tumor-Promoting Phorbol Ester Causes a Large Increase in APOBEC3A Expression and a Moderate Increase in APOBEC3B Expression in a Normal Human Keratinocyte Cell Line without Increasing Genomic Uracils

“…We recently described assays to quantify genomic uracils using alkoxyamines ARP and AA3 (41, 42). These chemicals react with the abasic sites created by the excision of uracils in DNA by the Ung and are then tagged with a fluorescent label for quantification (41,42). More recently, we also described another alkoxyamine, AA6, which also reacts with abasic sites and can be attached with a fluorescent tag using copper-free click chemistry ( Fig.…”

“…The uracils in the genomic DNA were quantified using an assay based on AA6, an alkoxyamine that can be coupled with a fluorescent dye using copper-free click chemistry (43). This assay is a modification of previously described assay using an alkoxyamine called AA3 (41). Briefly, HaeIII-digested genomic DNA was incubated with methoxyamine (Fisher Scientific) at a 10 mM final concentration to block the endogenous abasic sites.…”

A novel class of chemicals that react with abasic sites in DNA and specifically kill B cell cancers

“…It should be noted that our assays are expected to detect all aldehydic lesions in DNA. These include intact AP sites, AP sites cleaved by an AP endonuclease or other enzymes, the formamido forms of a number of oxidation and alkylation-induced adducts [ 24 ], and we use the phrase “AP site quantification” for textual convenience. Quantification of AP sites using AA3 was performed as described previously [ 24 ].…”