Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A⅐oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases have been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2-deoxyadenosine in which a single 2-H atom was replaced by fluorine. The structure of this fluorinated lesionrecognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.base-excision repair ͉ general acid catalysis ͉ substrate recognition T he genotoxic DNA lesion 8-oxoguanine (oxoG) arises chronically in cells through the attack of endogenous electrophilic oxidants on guanine residues (Fig. 1A). Left unrepaired, these oxoG lesions mispair with A during DNA replication, thereby giving rise to G⅐C to T⅐A transversion mutations. Most organisms possess a repair system dedicated to countering the deleterious effects of oxoG. The so-called GO system in eubacteria (1, 2) (Fig. 1B), for example, consists of proteins that directly sanitize the nucleotide precursor pool (MutT) and DNA (MutM or Fpg) of oxoG residues. Proteins that serve functions equivalent to those of MutT and MutM are found in all eukaryotic organisms (3). Failure of this first round of defense results in replicative production of oxoG:A pairs (4), which are particularly troublesome to repair, with neither nucleobase providing faithful information with which to direct correction of the other: oxoG does not belong in DNA at all, and although A is a canonical nucleobase, it is contextually aberrant in the oxoG:A pair because it replaces what should be a C. The multistep repair of oxoG:A is initiated by the highly evolutionarily conserved enzyme adenine DNA glycosylase (MutY in bacteria, hMYH in humans), which catalyzes hydrolysis of the glycosidic linkage between th...

To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β (polβ), we report four crystal structures of polβ complexed with dG•dTTP and dA•dCTP mismatches in the presence of Mg2+ or Mn2+. The Mg2+-bound ground-state structures show that the dA•dCTP-Mg2+ complex adopts an ‘intermediate’ protein conformation while the dG•dTTP-Mg2+ complex adopts an open protein conformation. The Mn2+-bound ‘pre-chemistry-state’ structures show that the dA•dCTP-Mn2+ complex is structurally very similar to the dA•dCTP-Mg2+ complex, whereas the dG•dTTP-Mn2+ complex undergoes a large-scale conformational change to adopt a Watson–Crick-like dG•dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that polβ increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, polβ appears to allow only a Watson–Crick-like conformation for purine•pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson–Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of polβ.

N7-Alkyl-2′-deoxyguanosines are major adducts in DNA that are generated by various alkylating mutagens and drugs. However, the effect of the N7-alkylation on the hydrogen bonding patterns of the guanine remains poorly understood. We prepared N7-methyl-2′-deoxyguanosine (N7mdG)-containing DNA using a transition-state destabilization strategy, developed a novel polβ-host-guest-complex system and determined eight crystal structures of N7mdG or dG paired with dC, dT, dG, and dA. The structures of N7mdG:dC and N7mdG:dG are very similar to those of dG:dC and dG:dG, respectively, indicating the involvement of the keto tautomeric form of N7mdG in the base pairings with dC and dG. On the other hand, the structure of N7mdG:dT shows that the mispair forms three hydrogen bonds and adopts a Watson-Crick-like geometry rather than a wobble geometry, suggesting that the enol tautomeric form of N7mdG involves in its base pairing with dT. In addition, N7mdG:dA adopts a novel shifted anti:syn base pair presumably via the enol tautomeric form of N7mdG. The polβ-host-guest-complex structures reveal that guanine-N7 methylation changes the hydrogen bonding patterns of the guanine when paired with dT or dA and suggest that N7 alkylation may alter the base pairing patterns of guanine by promoting the formation of the rare enol tautomeric form of guanine.

No abstract

The predominant product of aberrant DNA methylation is the genotoxic lesion N7-methyl-2'-deoxyguanosine (m7dG). M7dG is recognized and excised by lesion-specific DNA glycosylases, namely AlkA in E. coli and Aag in humans. Structural studies of m7dG recognition and catalysis by these enzymes have been hampered due to a lack of efficient means by which to incorporate the chemically labile m7dG moiety site-specifically into DNA on a preparative scale. Here we report a solution to this problem. We stabilized the lesion toward acid-catalyzed and glycosylase-catalyzed depurination by 2'-fluorination and toward base-catalyzed degradation using mild, nonaqueous conditions in the DNA deprotection reaction. Duplex DNA containing 2'-fluoro-m7dG (Fm7dG) cocrystallized with AlkA as a host-guest complex in which the lesion-containing segment of DNA was nearly devoid of protein contacts, thus enabling the first direct visualization of the N7-methylguanine lesion nucleobase in DNA. The structure reveals that the base-pairing mode of Fm7dG:C is nearly identical to that of G:C, and Fm7dG does not induce any apparent structural disturbance of the duplex structure. These observations suggest that AlkA and Aag must perform a structurally invasive interrogation of DNA in order to detect the presence of intrahelical m7dG lesions.