A method for detecting abasic sites in living cells: Age-dependent changes in base excision repair

Abstract

DNA damage ͉ aging ͉ glycosylase ͉ AP site A purinic and apyrimidinic (AP or abasic) sites are common lesions in DNA and are formed either spontaneously or as intermediates during the course of base excision repair (BER) of oxidized, deaminated, or alkylated bases (1, 2). N-glycosylases of BER recognize and excise the aberrant bases from DNA, resulting in formation of an AP site. The AP site is then incised by an AP endonuclease to generate a 5Ј-terminal base-free deoxyribose phosphate at a single-strand break. The repair of the single-strand break (AP site) is accomplished by a DNA polymerase ␤ (␤-pol) and DNA ligase (ligase I or III͞XRCC1) to restore the normal and correct base (3, 4). The rate-limiting step of BER is the lyase activity associated with the 8-kDa domain of ␤-pol, which removes the AP site from DNA (4). AP sites can be mutagenic (5-7) or can cause cell death (8-10).The methods (11-14) that detect AP sites after DNA isolation, including those with ARP (15, 16), may be inaccurate as a result of several factors: (i) artifactual AP sites resulting from base loss and formation of AP sites in DNA by high temperature at neutral pH (12, 17, 18); (ii) AP sites in DNA are subject to loss by ␤-elimination, followed by ␦-elimination catalyzed by high temperatures (19), primary amines in histones, polyamines (19-21), and thiols (22), causing underestimation of AP levels in DNA. Nakamura et al. (23) studied the endogenous level of AP sites in different tissues of the rat using aldehyde-reactive probe (ARP), isolating the DNA before trapping AP sites. They estimate that there are 50,000-200,000 AP sites͞mammalian cell, that the brain of rat contained the most AP sites, and that most of the AP sites were cleaved 5Ј to the AP sites. The first two results are different from the results reported here.In this paper, we describe a method for biotinylating AP sites in live cells and then quantifying the biotin in the isolated DNA. This method is used to determine the age-dependent changes in BER in human fibroblasts (IMR90), leukocytes isolated from human blood, and nuclei isolated from rat tissues. Materials and Methods Materials.A QIAamp blood kit was purchased from Qiagen (Chatsworth, CA). The DNA-precipitating agent, DAPER [N,NЈ-bis(3,3Ј-(dimethyl-amino)propylamine)-3,4,9,10-perylenetetra-carboxylicdiimide] and ImmunoPure 3,5,3Ј,5Ј-tetramethylbenzidine horseradish peroxidase (HRP) substrate kits were from Pierce. Vectastain ABC kit was from Vector Laboratories. O-Methoxyamine, EGTA, and Histopaque 1077 were from Sigma. HK-UNG thermolabile uracil N-glycosylase (UNG) was from Epicentre Technologies (Madison, WI). Hydrogen peroxide (30%) and Tween 20 were from Fisher. Fatty acid-free BSA was from Calbiochem. Rnase A was from Roche Molecular Biochemicals. Calf thymus DNA was from Worthington. ARP NЈ-aminooxy-methylcarbonyl hydrazino-D-biotin, was from Dujindo Laboratories (Kumamoto, Japan). Methyl methanesulfonate (MMS) and 1-octane sulfonic acid were from Fluka.Preparation of Nuclear Fraction from Rat Liver and Bra...

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

“…We calculate that 1.22% uracil by weight is equivalent to 12.2 μg uracil per mg of DNA, 14% replacement of T by U, or ~35,000 U/10 6 nt. In a subsequent report, Atamna et al [30] determined by GC/MS that the amount of uracil in CJ236 DNA was actually 0.28%, which we calculate to be ~8000 U/10 6 nt. The effect of temperature on dUMP incorporation in an E. coli dut ung strain was investigated by Warner and co-workers, who propagated BD1157 (dut-1 ung-1) at different temperatures in the presence of [6-3 H]uridine [8].…”

“…In some cases, uracil-DNA glycosylase was used to selectively excise the uracil base from the DNA and create a uracil-excision-mediated apyrimidinic site. In order to monitor uracil excision, HPLC-tandem mass spectrometry [29] and GC/MS [30] techniques were developed to measure the free uracil released following the reaction with Ung. An alternative approach was developed based on the detection of the uracil-excisionmediated AP-site using covalent modification with [ 14 C]methoxyamine [31].…”

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene

“…The actual levels of AP sites in cells, however, appear to be higher (18 000 ± 200 000 AP sites per cell per day, depending on the tissue and age) (Nakamura and Swenberg, 1999;Atamna et al, 2000). Thus, there could be tissue heterogeneity of BER.…”

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

“…This has been most apparent for DNA biomarkers with oxidative artifacts that both generate 8-oxo-dG (39) and consume it (5), problems that are avoided by DNA isolation from cells using the sodium iodide chaotropic method (43), manipulation of DNA in Chelex-treated buffers (43), addition of antioxidants such as desferal (43,44) or TEMPO (2,2,6,6-tetramethylperidinoxyl) (45).…”