DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5′-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase β (Pol β) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol β activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.Isolated damaged nucleotides are believed to be the most common lesions resulting from DNA oxidation. As a result of efforts by many researchers, a great deal has been learned about how DNA lesions are repaired by base excision repair (BER) 1 and nucleotide excision repair (NER) (1-3). Recently, clusters of DNA lesions have been identified as significant components of radiation-induced damage. Clustered lesions consist of two or more damaged nucleotides within ~1.5 turns of duplex DNA and are attributed to multiple ionization events by a single radiation track. In principle, clustered lesions can contain modified nucleotides on a single strand, but biochemical studies have focused on bistranded lesions, which are possible progenitors to double strand breaks (1-11). Numerous studies have revealed the effects of clustered lesions on BER. The effects are dependent upon the nature of the lesions, as well as their proximity to one another in duplex DNA. Clustered lesions consisting of two contiguously damaged nucleotides are referred to as tandem lesions (12,13). Unlike other examples of clustered damage, tandem lesions can result from a single DNA damaging event, in which a † We are grateful for support of this research by the National Institute of General Medical Science (Grant GM-063028). This research was supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.* To whom correspondence should be addressed. . E-mail: mgreenberg@jhu.edu. ‡ These researchers contributed equally to the research described in this paper. § Johns Hopkins University. || Laboratory of Molecular Genetics, NIEHS, NIH.1 Abbreviations: AP, abasic site; L, 2-deoxyribonolactone; F, tetrahydrofuran abasic site; Tg, thymine glycol; BER, base excision repair; LP-BER, long-patch base exc...
Reactive oxygen species (ROS) and serum ferritin levels are both considered to be important biological factors in the pathogenesis of myelodysplastic syndrome (MDS). This study evaluated the levels of ROS in 40 patients with MDS (19 males and 21 females) using the Free Radical Analytical System, FRAS4, and derivatives of reactive oxygen metabolite kits. The patients' mean age was 67.3 years (range 58 - 86 years). The sera of 34 (85%) patients exhibited higher levels of oxidative stress than the reference range. There was a positive correlation between ROS levels and serum ferritin levels, and a negative correlation between ROS levels and haemoglobin levels. There was a negative relationship between serum haemoglobin and ferritin levels. The results indicated that iron accumulation or severe anaemia could contribute to oxidative stress in MDS patients. Iron chelation and antioxidant therapy may be suitable for the management of MDS.
The nucleoside triphosphates of N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP. Similarly, polymerase-mediated introduction of beta-C-Fapy.dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates.
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