The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress. A recent report suggests that this lesion forms interstrand cross-links. Using duplexes in which C4-AP is produced from a synthetic precursor, we show that the lesion produces interstrand cross-links in which both strands are in tact and cross-links in which the C4-AP containing strand is cleaved. The yields of these products are dependent upon the surrounding nucleotide sequence. When C4-AP is opposed by dA, cross-link formation occurs exclusively with an adjacent dA on the 5'-side. Moreover, formation of the lower molecular weight cross-link is promoted by an opposing adenine. When the opposing dA is replaced by dT, the activity of the adenine can be rescued by adding the free base. This is a rare example in which DNA promotes its own modification, an observation that is all the more important because of the biological significance of the product produced.

The C4'-oxidized abasic site (C4-AP) is a commonly formed DNA lesion, which generates two types of interstrand cross-links (ICLs). The kinetically favored cross-link consists of two full length strands and forms reversibly and exclusively with dA. Cross-link formation is attributed to condensation of C4-AP with the N6-amino group of dA. Formation of the thermodynamic ICL involves cleavage of strand containing C4-AP on the 3'-side of the lesion. The ratios and yields of the ICLs are highly dependent upon the local sequence. Product analysis of enzyme digested material reveals that the ICL with dA is a cyclic adduct. Formation of the thermodynamically favored cross-link is catalyzed by the surrounding DNA sequence, and occurs favorably with dC and dA, but not with dG or dT. Mechanistic studies indicate that β-elimination from C4-AP is the rate limiting step in the formation of the thermodynamic ICL and that the local DNA environment determines the rate constant for this reaction. The efficiency of ICL formation, the stability of the thermodynamic product, and their possible formation in cells (Regulus, P.; et al. Proc. Nat. Acad. Sci. USA 2007, 104, 14032-14037.) suggest that this lesion will be deleterious to the biological system in which they are produced. KeywordsDNA damage; reaction mechanism; interstrand cross-link DNA interstrand cross-links block replication and transcription. Consequently, they are associated with the cytotoxic effects of chemotherapeutic agents that are bis-alkylators, such as the mitomycins and nitrogen mustards. [1][2][3][4][5] Purposeful interstrand cross-linking is also useful as a tool for probing nucleic acid structure and in biotechnology. [6][7][8][9][10][11][12][13] More recent investigations implicate endogenously produced bis-alkylating agents resulting from lipid and DNA oxidation in ICL formation. 14 In other instances, environmental pollutants and/or their metabolites have been found to produce ICLs. 15,16 Each of these examples involves covalent linkage of the opposing DNA strands by a bridging molecule. The formation of ICLs by direct reaction of one nucleic acid strand with its hybridization partner was firmly established in 2005. In one instance a DNA radical generated by reaction with hydroxyl radical produced ICLs with the opposing dA. [17][18][19] The Gates group was the first to unequivocally describe the reaction of an abasic site with one or more nucleotides on the opposing strand. 20 They showed that the ubiquitous abasic site (AP) reacts with the deoxyguanosine opposite the 5'-flanking dC. We recently reported that the C4'-oxidized abasic site (C4-AP) also produces ICLs. 21 In this manuscript we elaborate on the scope of this reaction and explore its mechanism. mgreenberg@jhu.edu,. Supporting Information Available. Complete description of experimental procedures, ESI-MS of oligonucleotides containing 6, sample autoradiograms, NMR spectra of 9. This material is available free of charge via the Internet at http://pubs.acs.org. C4-AP is a major product of...

RNA oxidation is important in the etiology of disease and as a tool for studying the structure and folding kinetics of this biopolymer. Nucleobase radicals are the major family of reactive intermediates produced in RNA exposed to diffusible species such as hydroxyl radical. The nucleobase radicals are believed to produce direct strand breaks by abstracting hydrogen atoms from their own and neighboring ribose rings. By independently generating the formal C5-hydrogen atom addition product of uridine in RNA, we provide the first chemical characterization of the pathway for direct strand scission from a RNA nucleobase radical. The process is more efficient under anaerobic conditions. The preference for strand scission in double-stranded RNA over single-stranded RNA suggests that this chemistry may be useful for analyzing the secondary structure of RNA in hydroxyl radical cleavage experiments if they are carried out under anaerobic conditions.

Nucleobase radicals are the major reactive intermediates produced when hydroxyl radical reacts with nucleic acids. 5,6-Dihydrouridin-6-yl radical (1) was independently generated from a ketone precursor via Norrish Type I photocleavage in a dinucleotide, single stranded, and double stranded RNA. This radical is a model of the major hydroxyl radical adduct of uridine. Tandem lesions resulting from addition of the peroxyl radical derived from 1 to the 5′-adjacent nucleotide are observed by ESI- MS. Radical 1 produces direct strand breaks at the 5′-adjacent nucleotide and at the initial site of generation. The preference for cleavage at these two positions depends upon the secondary structure of the RNA and whether O2 is present or not. Varying the identity of the 5′-adjacent nucleotide has little effect on strand scission. In general, strand scission is significantly more efficient under anaerobic conditions than when O2 is present. Strand scission is more than twice as efficient in double stranded RNA than in a single stranded oligonucleotide under anaerobic conditions. Internucleotidyl strand scission occurs via β-fragmentation following C2′-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical ultimately yields products containing 3′-phosphate or 3′-deoxy-2′-ketouridine termini. These end groups are proposed to result from competing deprotonation pathways. The dependence of strand scission efficiency from 1 on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions.

The C4′-oxidized abasic site (C4-AP), which is produced by a variety of damaging agents has significant consequences on DNA. The lesion is highly mutagenic and reactive, resulting in interstrand cross-links. The base excision repair of DNA containing independently generated C4-AP was examined. C4-AP is incised by Ape1 ~12-fold less efficiently than an apurinic/ apyrimidinic lesion. DNA polymerase β induces the β-elimination of incised C4-AP in ternary complexes, duplexes, and single stranded substrate. However, excision from a ternary complex is most rapid. In addition, the lesion inactivates the enzyme after ~7 turnovers on average by reacting with one or more lysine residues in the lyase active site. Unlike 5′-(2-phosphoryl-1,4-dioxobutane) which very efficiently irreversibly inhibits Pol β, the lesion is readily removed by strand displacement synthesis carried out by the polymerase in conjunction with flap endonuclease 1. DNA repair inhibition by C4-AP may be a partial cause of the cytotoxicity of drugs that produce this lesion. KeywordsDNA damage; base excision repair; inhibition; oxidized abasic site Hydrogen atom abstraction from the 2′-deoxyribose rings of nucleotides in DNA gives rise to several oxidized abasic lesions (1,2). One of these, the C4′-oxidized abasic site (C4-AP, Chart 1) is produced by a variety of DNA damaging agents, including γ-radiolysis and antitumor antibiotics (3-5). Its frequent occurrence is attributed to the high accessibility of the C4′-hydrogen atom to diffusible species, and the relatively low bond dissociation energy of the respective carbon-hydrogen bond (6,7). C4-AP is efficiently incised by the endonucleases in E. coli that are responsible for AP incision (8). In addition, previous studies using C4-AP produced by bleomycin indicated that the lesion is a substrate for mammalian BER enzymes, including Ape1 and Pol β (9). Ape1 incision and subsequent Pol β excision are the first two steps in BER of AP lesions (Scheme 1). However, this pathway does not efficiently excise all abasic lesions. DOB is an oxidized abasic lesion that is produced by a variety of DNA damaging agents. Recent experiments revealed that DOB very efficiently inhibits DNA polymerase β irreversibly (10). C4-AP contains the 1,4-dicarbonyl functional group that is crucial for irreversible Pol β inhibition by DOB. The structural similarity between these two lesions, the central role played by Pol β in BER, and