Repair of endogenous DNA base lesions modulate lifespan in mice.

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O(6)-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse.

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents.

O6-Methylguanine DNA lesions induce an intra-S-phase arrest from which cells exit into apoptosis governed by early and late multi-pathway signaling network activation.

The O(6)-methylguanine (O(6)MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O(6)MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O(6)MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O(6)MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment.

Frameshift mutagenesis and microsatellite instability induced by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine, and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than does the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L-induced mutator phenotype.

Aag-initiated base excision repair drives alkylation-induced retinal degeneration in mice.

Vision loss affects >3 million Americans and many more people worldwide. Although predisposing genes have been identified their link to known environmental factors is unclear. In wild-type animals DNA alkylating agents induce photoreceptor apoptosis and severe retinal degeneration.

Substrate binding pocket residues of human alkyladenine-DNA glycosylase critical for methylating agent survival.

Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination.

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis.

Nucleotide excision repair defect influences lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent in the absence of base excision repair.

Using a yeast shuttle vector system, we have previously reported on the toxicity and mutagenicity of Me-lex, [1-methyl-4-[1-methyl-4-[3-(methoxysulfonyl)propanamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propane, a compound that selectively generates 3-methyladenine (3-MeA). We observed that a mutant strain defective in Mag1, the glycosylase that excises 3-MeA in the initial step of base excision repair (BER) to generate an abasic site, is significantly more sensitive to the toxicity of Me-lex with respect to wild type but shows only a marginal increase in mutagenicity. A strain defective in AP endonuclease activity (Deltaapn1apn2), also required for functional BER, is equally sensitive to the toxicity as the Deltamag1 mutant but showed a significantly higher mutation frequency.

DNA damage and cytotoxicity induced by minor groove binding methyl sulfonate esters.

Minor groove specific DNA equilibrium binding peptides (lex) based on N-methylpyrrole-carboxamide and/or N-methylimidazolecarboxamide subunits have been modified with an O-methyl sulfonate ester functionality to target DNA methylation in the minor groove at Ade/Thy- and/or Gua/Cyt-rich sequences. HPLC and sequencing gel analyses show that the Me-lex compounds all selectively react with DNA to afford N3-alkyladenine as a major adduct. The formation of the N3-alkyladenine lesions is sequence-dependent based on the equilibrium binding preferences of the different lex peptides.

Evidence in Escherichia coli that N3-methyladenine lesions and cytotoxicity induced by a minor groove binding methyl sulfonate ester can be modulated in vivo by netropsin.

The use of DNA equilibrium binding molecules to transfer alkyl groups to specific positions on DNA is an approach to generating cytotoxic DNA damage while avoiding the formation of promutagenic lesions that increase the risk for the development of secondary cancer. We have previously reported that in vitro a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (Me-lex) selectively affords N3-methyladenine lesions in >90% yield relative to the formation of other adducts. While in vitro interactions between the lex dipeptide and DNA have been thoroughly studied, in vivo interactions are more difficult to elucidate.

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent.

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1.