The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix (HhH) DNA glycosylase superfamily, excises adenine from mispairs with 8-oxoguanine and guanine. High-resolution crystal structures of the MutY catalytic core (cMutY), the complex with bound adenine, and designed mutants reveal the basis for adenine specificity and glycosyl bond cleavage chemistry. The two cMutY helical domains form a positively-charged groove with the adenine-specific pocket at their interface. The Watson-Crick hydrogen bond partners of the bound adenine are substituted by protein atoms, confirming a nucleotide flipping mechanism, and supporting a specific DNA binding orientation by MutY and structurally related DNA glycosylases.

The base excision repair pathway is an organism's primary defense against mutations induced by oxidative, alkylating, and other DNA-damaging agents. This pathway is initiated by DNA glycosylases that excise the damaged base by cleavage of the glycosidic bond between the base and the DNA sugar-phosphate backbone. A subset of glycosylases has an associated apurinic/apyrimidinic (AP) lyase activity that further processes the AP site to generate cleavage of the DNA phosphate backbone. Chemical mechanisms that are supported by biochemical and structural data have been proposed for several glycosylases and glycosylase/AP lyases. This review focuses on the chemical mechanisms of catalysis in the context of recent structural information, with emphasis on the catalytic residues and the active site conformations of several cocrystal structures of glycosylases with their substrate DNAs. Common structural motifs for DNA binding and damage specificity as well as conservation of acidic residues and amino groups for catalysis are discussed.

Endogenously formed reactive oxygen species continuously damage cellular constituents including DNA. These challenges, coupled with exogenous exposure to agents that generate reactive oxygen species, are both associated with normal aging processes and linked to cardiovascular disease, cancer, cataract formation, and fatty liver disease. Although not all of these diseases have been definitively shown to originate from mutations in nuclear DNA or mitochondrial DNA, repair of oxidized, saturated, and ring-fragmented bases via the base excision repair pathway is known to be critical for maintaining genomic stability. One enzyme that initiates base excision repair of ring-fragmented purines and some saturated pyrimidines is NEIL1, a mammalian homolog to Escherichia coli endonuclease VIII. To investigate the organismal consequences of a deficiency in NEIL1, a knockout mouse model was created. In the absence of exogenous oxidative stress, neil1 knockout (neil1 ؊/؊ ) and heterozygotic (neil1 ؉/؊ ) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia. In humans, this combination of clinical manifestations, including hypertension, is known as the metabolic syndrome and is estimated to affect >40 million people in the United States. Additionally, mitochondrial DNA from neil1 ؊/؊ mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. These data suggest an important role for NEIL1 in the prevention of the diseases associated with the metabolic syndrome.DNA repair ͉ fatty liver disease ͉ mitochondria ͉ obesity ͉ oxidative stress A fter exposure to reactive oxygen species (ROS), the major purine lesions are 8-oxoguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine (1-5). To reverse the potentially deleterious effects of oxidative DNA base lesions, cells primarily use the base excision repair (BER) pathway to restore the DNA to its undamaged state (6). The BER pathway is initiated by lesion-specific DNA glycosylases that catalyze bond scission and release the damaged base from the deoxyribose sugar.Unlike nucleotide excision repair, which functions only on nuclear DNA, the BER pathway is operative on both nuclear DNA and mitochondrial DNA (mtDNA). Although BER in the mitochondria repairs normal endogenous oxidant-induced DNA damage, expression of mitochondrially targeted DNA glycosylases that are specific for the repair of oxidatively induced DNA lesions leads to enhanced repair and increased survival after ROS challenge (7-10). These data emphasize that maintenance of the mitochondrial genome is a delicate balance of mtDNA copy number and BER capacity.To initiate repair of oxidative lesions, mammalian cells primarily use the products of the ogg1, nth1, neil1, and neil2 genes (11). These corresponding proteins have somewhat overlapping substrate specificities that may explain, at least partially, the absence of obvious phenotypes in ogg1-and nth1-null mice. NEIL1 has a strong sub...

Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N 2 -guanines, similar to crosslinks formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ϳ97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 and 3 ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol or a pol /Rev1 combination. Because pol was able to bypass these ICLs, biological evidence for a role for pol in tolerating the N 2 -N 2 -guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol -depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol in the processing of N 2 -N 2 -guanine ICLs.

The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses indicate that the DNA interstand cross-links contain a mixture of carbinolamine and Schiff base, with the carbinolamine forms of the linkages predominating in duplex DNA. The reduced derivatives of the enal-mediated N2-dG:N2-dG interstrand cross-links can be processed in mammalian cells by a mechanism not requiring homologous recombination. Mutations are rarely generated during processing of these cross-links. In contrast, the reduced acrolein-mediated N2-dG peptide conjugates can be more mutagenic than the corresponding monoadduct. DNA polymerases of the DinB family, pol IV in E. coli and pol κ in human, are implicated in error-free bypass of model acrolein-mediated N2-dG secondary adducts, the interstrand cross-links, and the peptide conjugates.