Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein crosslinks and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.chromatin | oxidative damage | histone modification E xogenously and endogenously produced agents continuously damage DNA. More than 70 types of damage have been identified, and significant effort is expended to determine which of these lesions are biologically important (1, 2). The apurinic/apyrimidinic (AP) site is a ubiquitous form of DNA damage that is produced in excess of 10,000 lesions per cell each day (3). This highly mutagenic lesion results from spontaneous hydrolysis of native and damaged nucleotides. AP sites are also formed as intermediates during base excision repair of alkylated and oxidized nucleotides and are themselves removed by multiple enzymatic pathways (4, 5). In mammalian cells incision of the AP's 5′-phosphate by Ape1, followed by excision of the resulting 5′-terminal 2′-deoxyribose phosphate by DNA polymerase β is the major pathway for removing the lesion. Redundant repair pathways are reflective of the high mutagenic potential and large quantities of AP produced and are indicative of their physiological significance. Mammalian cells lacking Ape1, the major base excision repair protein responsible for incising AP sites, are embryonic lethal (6). Recent observations reinforce the perception that AP sites are cytotoxic. For instance, formation of bursts of AP sites by the natural product leinamycin is postulated to be the source of this antitumor agent's cy...

Duplex DNA containing an apurinic/apyrimidinic (AP) lesion undergoes cleavage significantly more rapidly in nucleosome core particles (NCPs) than it does when free. The mechanism of AP cleavage within NCPs was studied through independently generating lesions within them. AP mediated DNA cleavage within NCPs is initiated by DNA-protein crosslink (DPCun) formation followed by β-elimination to give DPCs containing cleaved DNA (DPCcl). Hydrolysis of DPCcl produces a DNA single strand break (SSB). C2-dideuteration of AP showed that deprotonation from this position is involved in the rate determining step. Experiments utilizing NCPs containing mutated histone H4 proteins indicated that lysine residues in the amino terminal tail are involved in both DPC formation and β-elimination steps. Lysines 16 and 20 seem to play a greater role in reacting with AP at superhelical location 1.5 but other amino acids (e.g. lysines 5, 8, and 12) compensate in their absence. The mechanism of rapid double strand breaks in bistranded, clustered AP lesions was studied by independently preparing reaction intermediates within model NCPs. A single strand break on one strand enhances the cleavage of a proximal AP on the opposite strand.

The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylasedeficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions.A large number of DNA base modifications are formed by oxidative damage to DNA (for review, see Ref. 1). Some of these lesions are generated at high rates, even in the absence of exogenous DNA-damaging agents. For instance, it was estimated that 100 -500 8-hydroxyguanines 3 are formed per day in a human cell (2). 8-oxoG is one of the most studied DNA lesions, and it is often used as a biomarker of oxidative DNA damage. However, ring-opened formamidopyrimidine lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA), are formed at equal or higher levels than 8-oxoG after oxidative stress (3-5). These lesions result from hydroxyl radical attack on guanine and adenine, respectively, followed by one-electron reduction of the hydroxyl adduct radicals (1), which are also intermediates in the formation of 8-oxoG and 8-oxoA (6). Formation of Fapy lesions in DNA upon UV radiation has also been reported (7). FapyA (8) and FapyG (9) are miscoding in vitro, both directing the preferential misincorporation of adenine opposite the lesions by a bacterial DNA polymerase (Klenow exo Ϫ ). Experiments using the methylated analogue of FapyG, i.e. 2,6-diamino-4-hydroxy-5-Nmethylformamidopyrimidine (Me-FapyG), have suggested that formamidopyrimidines might also constitute blocks to DNA poly...

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.

Oxidized abasic residues in DNA constitute a major class of radiation and oxidative damage. Free radical attack on the nucleotidyl C-1 carbon yields 2-deoxyribonolactone (dL) as a significant lesion. Although dL residues are efficiently incised by the main human abasic endonuclease enzyme Ape1, we show here that subsequent excision by human DNA polymerase ␤ is impaired at dL compared with unmodified abasic sites. This inhibition is accompanied by accumulation of a protein-DNA cross-link not observed in reactions of polymerase ␤ with unmodified abasic sites, although a similar form can be trapped by reduction with sodium borohydride. The formation of the stably cross-linked species with dL depends on the polymerase lysine 72 residue, which forms a Schiff base with the C-1 aldehyde during excision of an unmodified abasic site. In the case of a dL residue, attack on the lactone C-1 by lysine 72 proceeds more slowly and evidently produces an amide linkage, which resists further processing. Consequently dL residues may not be readily repaired by "shortpatch" base excision repair but instead function as suicide substrates in the formation of protein-DNA crosslinks that may require alternative modes of repair.Mutagenesis and disruption of the cell cycle caused by DNA damage is counteracted by DNA repair systems. In the base excision repair pathway (1-3), DNA glycosylases eliminate damaged bases to generate abasic (AP) 1 sites, which are also formed in large numbers by spontaneous depurination (2). In either case, AP sites are incised by an AP endonuclease to allow subsequent DNA repair synthesis and excision of the abasic residue. In mammalian cells, incision is carried out by the major AP endonuclease Ape1 protein (also called Apex, Hap1, or Ref1), while the excision step for regular abasic residues is thought to be mainly carried out by DNA polymerase ␤ (Pol␤) using a ␤-elimination mechanism. A distinct branch of the base excision pathway involves strand displacement repair synthesis and excision of the displaced, damaged strand by the FEN1 nuclease (4 -6). Still another variation is potentiated by the initial DNA glycosylase (7) because some of these enzymes carry out a second reaction to cleave at the abasic site by ␤- elimination (1, 3). The resulting 3Ј-blocked products must then be removed by an enzyme such as Ape1 before repair synthesis can proceed (1).Base excision repair acts on a wide variety of deaminated, alkylated, or oxidized bases (2, 3). However, oxidative damage to DNA also produces various modified abasic residues that may complicate the repair scenario (1). For example, free radical attack forms strand breaks with fragmentary or oxidized products of deoxyribose; when these are present at the 3Ј terminus, removal by Ape1 may be the rate-limiting repair step (8, 9). Oxidized abasic residues without direct strand breakage (10) include 2-deoxypentos-4-ulose residues (a major lesion produced by the antitumor drug bleomycin) and 2-deoxyribonolactone (dL) residues (formed by diverse oxidative agents). 2-D...

Nucleobase radicals (e.g., 1) are the major family of reactive intermediates formed when DNA is exposed to gamma-radiolysis. Independent generation of 1 in chemically synthesized oligonucleotides reveals that formation of this nucleobase radical under aerobic conditions results in the formation of tandem lesions approximately 65% of the time. The distribution of lesions formed with the 5'- and 3'-adjacent nucleotides is dependent upon the secondary structure of duplex DNA. Tandem lesions, which are defined as two contiguously, damaged nucleotides in a single DNA strand, are of significant biological interest. The yield of tandem lesions from 1 is much greater than was previously believed. The observations presented could have significant ramifications on how scientists interpret the effects of gamma-radiolysis on DNA.

A 5-(2′-Deoxyuridinyl)methyl radical (1) was independently generated from three photochemical precursors and is the first example of a DNA radical that forms interstrand cross-links. Oxygen labeling experiments support generation of 1 by all precursors. Interstrand cross-links are produced upon irradiation of DNA containing any of the precursors. Cross-linking occurs via reaction with the opposing 2′-deoxyadenosine and is independent of O 2 . The independence of cross-link formation on O 2 is explained by kinetic analysis, which shows that the radical reacts reversibly with O 2 . Examination of the effects of glutathione on cross-link formation under anaerobic conditions suggests that adoption of the syn-conformation by 1 is the rate-limiting step in the process. Interstrand crosslink formation is reversible in the presence of a good nucleophile. The stability of the interstrand cross-link suggests that the isolated molecule is a rearrangement product of that formed in solution. The rearrangement is a consequence of the isolation procedure but also occurs slowly in solution. Oxygen independent cross-link formation may be useful for the purposeful damage of DNA in hypoxic tumor cells, where O 2 is deficient.The biological importance of DNA damage is evident in many ways. The double sword nature of these chemical transformations is illustrated by their involvement in the etiology and treatment of cancer. 1-3 In addition, there is a growing appreciation of the importance of DNA damage in aging. 4-6 DNA radicals are an important family of reactive intermediates that give rise to modified intact polymers and also lead to single-and double-strand breaks. 7-9 Recently, we reported that an independently generated 5-(2′-deoxyuridinyl)methyl radical (1) produced interstrand cross-links (ISCs) with an opposing 2′-deoxyadenosine (dA) (Scheme 1). 10 The radical is derived from formal hydrogen atom abstraction from the methyl group of thymidine and is not believed to be formed in large amounts by ionizing radiation despite the low bond dissociation energy (BDE) of the carbon-hydrogen bonds and their accessibility in the major groove. The preliminary report concerning 1 was the first explicit example of DNA interstrand cross-link (ISC) formation through a nucleic acid radical. ISCs are often responsible for the cytotoxic effects of DNA damaging agents. 11,12 Hence, ISC formation by a radical produced in even small amounts (e.g., 1) by agents such as γ-radiolysis could be biologically significant. Our interest in the radical-mediated cross-link formation is enhanced by the observation that the process involving 1 is independent of O 2 . This suggests that, in addition to being chemically novel, the formation of interstrand cross-links from 1 may be useful in hypoxic cells, such as those present in tumors. In addition to arising from direct hydrogen atom abstraction, the 5-(2′-deoxyuridinyl)methyl radical (1) can be formed from a two-step process, such as that induced by the direct effect of γ-radiolysis (Scheme 1). In a...

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...