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DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase ␤ (␤-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of ␤-pol-mediated 5 -deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of ␤-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that ␤-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery.

Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision-repair enzymes, AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology.

The human 3-methyladenine DNA glycosylase (AAG) recognizes and excises a broad range of purines damaged by alkylation and oxidative damage, including 3-methyladenine, 7-methylguanine, hypoxanthine (Hx), and 1,N 6 -ethenoadenine (εA). The crystal structures of AAG bound to εA have provided insights into the structural basis for substrate recognition, base excision, and exclusion of normal purines and pyrimidines from its substrate recognition pocket. In the present study, we explored the substrate specificity of full-length and truncated Δ80AAG on a library of oligonucleotides containing structurally diverse base modifications. Substrate binding and base excision kinetics of AAG with 13 damaged oligonucleotides were examined. We found that AAG bound to a wide variety of purine and pyrimidine lesions, but excised only few of them. Singleturnover excision kinetics showed that in addition to the well-known εA and Hx substrates, 1-methylguanine (m1G) was also excised efficiently by AAG. Thus, along with εA and ethanoadenine (EA), m1G is another substrate that is shared between AAG and the direct repair protein AlkB. In addition, we found that both the full-length and truncated AAG excised 1,N 2 -ethenoguanine (1,N 2 -εG), albeit weakly, from duplex DNA. Uracil was excised from both single-and double-stranded DNA, but only by the full-length AAG, indicating that the N-terminus of AAG may influence glycosylase activity for some substrates. Although AAG has been primarily shown to act on doublestranded DNA, AAG excised both εA and Hx from single-stranded DNA, suggesting the possible significance of repair of these frequent lesions in single-stranded DNA transiently generated during replication and transcription.DNA damaging agents are ubiquitous and cellular DNA is constantly attacked by a variety of endogenous and exogenous DNA damaging agents. DNA can be deaminated spontaneously or alkylated by endogenous intracellular sources and by exogenous environmental agents. Such damages can interfere with DNA replication and transcription, and may be mutagenic or † This work was supported by NIH grants (ES05355, CA75576, CA55042, ES02109, T32-ES007020, CA80024, and CA26731) and NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cytotoxic to the cell. During evolution, multiple DNA repair pathways have evolved to maintain the integrity of DNA in all organisms. Among other pathways, single base aberrations can be repaired by the base excision repair (BER) pathway. BER is initiated by DNA glycosylases that recognize the damaged base in the genome, followed by hydrolysis of the N-glycosylic bond, resulting in the release of the damaged base and the generation of an abasic site. The abasic site is further processed by an AP endonuclease or AP lyase, resulting in a strand break. After trimming of the DNA ends, DNA is resynthesized by a DNA polymerase and a DNA ligase seals the nick to restore undamaged ...

The human 3-methyladenine DNA glycosylase (AAG) is a repair enzyme that removes a number of damaged bases from DNA, including adducts formed by some chemotherapeutic agents. Cisplatin is one of the most widely used anticancer drugs. Its success in killing tumor cells results from its ability to form DNA adducts and the cellular processes triggered by the presence of those adducts in DNA. Variations in tumor response to cisplatin may result from altered expression of cellular proteins that recognize cisplatin adducts. The present study focuses on the interaction between the cisplatin intrastrand cross-links and human AAG. Using site-specifically modified oligonucleotides containing each of the cisplatin intrastrand cross-links, we found that AAG readily recognized cisplatin adducts. The apparent dissociation constants for the 1, 2-d(GpG), the 1,2-d(ApG), and the 1,3-d(GpTpG) oligonucleotides were 115 nM, 71 nM, and 144 nM, respectively. For comparison, the apparent dissociation constant for an oligonucleotide containing a single 1,N(6)-ethenoadenine (epsilonA), which is repaired efficiently by AAG, was 26 nM. Despite the affinity of AAG for cisplatin adducts, AAG was not able to release any of these adducts from DNA. Furthermore, it was demonstrated that the presence of cisplatin adducts in the reactions inhibited the excision of epsilonA by AAG. These data suggest a previously unexplored dimension to the toxicological response of cells to cisplatin. We suggest that cisplatin adducts could titrate AAG away from its natural substrates, resulting in higher mutagenesis and/or cell death because of the persistence of AAG substrates in DNA.

DNA glycosylases initiate base excision repair by first binding, then excising aberrant DNA bases. S. cerevisiae encodes a 3-methyladenine (3MeA) DNA glycosylase, Mag, that recognizes 3MeA and various other DNA lesions including 1,N 6 -ethenoadenine (εA), hypoxanthine (Hx) and abasic (AP) sites. In the present study, we explore the relative substrate specificity of Mag for these lesions and in addition, show that Mag also recognizes cisplatin cross-linked adducts, but does not catalyze their excision. Through competition binding and activity studies, we show that in the context of a random DNA sequence Mag binds εA and AP-sites the most tightly, followed by the cross-linked 1,2-d(ApG) cisplatin adduct. While εA binding and excision by Mag was robust in this sequence context, binding and excision of Hx was extremely poor. We further studied the recognition of εA and Hx by Mag, when these lesions are present at different positions within A:T and G:C tracts. Overall, εA was slightly less well excised from each position within the A:T and G:C tracts compared to excision from the random sequence, whereas Hx excision was greatly increased in these sequence contexts (by up to 7-fold) compared to the random sequence. However, given most sequence contexts, Mag had a clear preference for εA relative to Hx, except in the TTXTT (X= εA or Hx) sequence context from which Mag removed both lesions with almost equal efficiency. We discuss how DNA sequence context affects base excision by various 3MeA DNA glycosylases.

Health. Because the degree of inflammation is dynamic and varies within the UC colon, two samples (paired tissues "X" and "Y") were taken from each surgical specimen. Thirty pairs of colon samples obtained during surgery from 30 patients with UC were available for analysis.Received for publication August 7, 2003, and accepted Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision-repair enzymes , AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology.