The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that 1,N 6 -ethenoadenine (⑀A) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O 6 -methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and ⑀A paired with T showed that excision of Hx was about four times faster than ⑀A, whereas binding assays showed that the binding affinity was about five times greater for ⑀A than for Hx. The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for ⑀A. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7-8. The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.The base excision repair pathway provides the cell with a major line of defense against damage to DNA bases by excising damaged bases and resynthesizing DNA. Base excision repair is initiated by the activity of DNA glycosylases, which function to identify and excise damaged bases. Because these enzymes recognize DNA base damage, they are key to the overall effectiveness of the pathway. Monofunctional DNA glycosylases such as human alkyladenine DNA glycosylase (hAAG) 1 excise damaged DNA bases by hydrolysis of the C1Ј-N glycosylic bond, forming a free DNA base and an abasic sugar residue. Once the damaged base is removed, other enzymes in the pathway remove the remaining sugar residue and resynthesize DNA to fill in the gap.DNA glycosylases are damage-specific; different enzymes are responsible for excising different types of damaged DNA bases. Some glycosylases such as uracil DNA glycosylase are very specific and excise only a single damaged base, uracil, in this case. Other DNA glycosylases have broader substrate specificities. For example, formamidopyrimidine DNA glycosylase (FaPy) recognizes oxidative damage to DNA bases and excises 7,8-dihydro-8-oxoguanine (8-oxoG), FaPy, and 5-hydroxycytosine. Based on both structural (for recent reviews, Refs. 1-3) and spectroscopic (4) data, DNA glycosylases are believed to "flip" damaged nucleotides out of the DNA helix and into an enzyme active site, where catalysis takes place. Given this type of flipping mechanism, it is easy to imagine how a DNA glycosylase may recognize a specific damaged DNA base through interactions in the active site that provide a "tight" fit and align the glycosylic bond for chemistry. For DNA glycosylases with broader substrate specificities, the nature of the structural interactions and me...

Human alkyladenine DNA glycosylase "flips" damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by "pushing" it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene, which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing 1,N 6 -ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine.Human alkyladenine DNA glycosylase (AAG) 1 is one of several damage-specific DNA glycosylases that function in the base excision repair pathway (reviewed in Refs. 1-4). These DNA glycosylases initiate repair by identifying and removing damaged bases from DNA. Monofunctional DNA glycosylases, including AAG, hydrolyze the glycosylic bond between the base and sugar to leave an abasic sugar residue in DNA. Other enzymes in the pathway remove this apurinic/apyrimidinic lesion and resynthesize DNA to complete repair. The ability of DNA glycosylases to identify and excise damaged DNA bases is key to the overall success of base excision repair.Structural studies of AAG (5, 6) and other DNA glycosylases have revealed that they use a nucleotide "flipping" mechanism for damaged base recognition and excision where the damaged base is flipped out of the DNA helix and bound in an enzyme active site. In these nucleotide-flipped DNA glycosylase⅐DNA complexes, an enzyme amino acid side chain is inserted into the base stack at the site vacated by the flipped base and may assist in nucleotide flipping by pushing the damaged base from the helix. It is believed that DNA glycosylases actively flip damaged bases out of the helix rather than passively capturing bases that have transiently adopted extrahelical conformations. This active nucleotide flipping mechanism is supported by detailed kinetic studies of Escherichia coli uracil DNA glycosylase which show a two-step binding mechanism where UDG initially binds DNA to form an unflipped protein⅐DNA complex prior to flipping uracil from the helix (7).Many questions remain about how nucleotide flipping enables DNA glycosylases to discriminate between damaged and undamaged bases. For DNA glycosylases that have a narrow substrate specificity, a mechanism where a...

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To investigate the effectiveness of EDL-291, a 6,7-dimethoxy-1-[4-(4-methoxypyridin-3-yl)benzyl]-1,2,3,4-tetrahydroisoquinoline dihydrochloride compound, in inhibiting the survival of glioblastoma in vitro and in vivo. Dose-response curves were generated to determine the EC50 in rat and human glioblastoma cell lines by treatment with different dilutions of EDL-291. To evaluate the architecture of the glioblastoma cells after treatment with EDL-291, the rat and human glioblastoma cells were stained with Mito Tracker Green FM. To determine whether autophagy was induced in EDL-291-treated glioblastoma cells, both rat and human glioblastoma cell lines were stained with acridine orange and light chain-3 immunoblots were performed. The efficacy of EDL-291 was monitored in vivo using a rat glioblastoma model. Rat glioblastoma cells were transplanted into an intracranial rat model, followed by infusions of saline, a low dose of EDL-291 (20 mg/kg for the first half hour, followed by 40 mg/kg EDL-291 in saline for 4 h), or a high dose of EDL-291 (60 mg/kg for the first half hour, followed by 90 mg/kg EDL-291 for 4 h). EDL-291 inhibits glioblastoma in vitro by destroying the mitochondria as shown with Mito Tracker Green FM. Acridine orange staining and light chain-3 immunoblots suggest that autophagy is induced when glioblastoma cells are treated with EDL-291. In vivo, a low dosage of EDL-291 is sufficient and effective in reducing glioblastoma tumor size. EDL-291 selectively induces cell death in rat and human glioblastoma cell lines by the induction of autophagy. EDL-291 exhibits antiglioblastoma effects both in vitro and in vivo.

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