Glycosylase Initiates Base Excision Repair Research Articles

NEIL1 and NEIL2 DNA glycosylases modulate anxiety and learning in a cooperative manner in mice

Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases affect cognition in mice, while the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in knockout mouse models. Neil1−/−Neil2−/− mice display hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels are comparable between genotypes and no mutator phenotype is found. Thus, impaired canonical repair is not likely to explain the altered behavior. Electrophysiology suggests reduced axonal activation in the hippocampal CA1 region in Neil1−/−Neil2−/− mice and lack of NEIL1 and NEIL2 causes dysregulation of genes in CA1 relevant for synaptic function. We postulate a cooperative function of NEIL1 and NEIL2 in genome regulation, beyond canonical BER, modulating behavior in mice.

Predicting Enhanced Cell Killing through PARP Inhibition

PARP inhibitors show promise as combination and single agents in cancer chemotherapy. Here, we evaluate results obtained with mouse fibroblasts and the common laboratory PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) and analyze the potential for enhanced cytotoxicity following the combination of a DNA-damaging agent and a PARP inhibitor. Methylated DNA bases are repaired by the monofunctional glycosylase-initiated single-nucleotide base excision repair (BER) pathway. An intermediate of this process has a single-nucleotide gap in double-stranded DNA containing the 5'-deoxyribose phosphate (dRP) group at one margin. This 5'-dRP group is removed by the lyase activity of pol β prior to gap filling; then completion of repair is by DNA ligation. PARP-1 binds to and is activated by the 5'-dRP group-containing intermediate, and poly(ADP-ribos)ylation is important for efficient repair. 4-AN-mediated sensitization to the methylating chemotherapeutic agent temozolomide is extreme, producing a level of cytotoxicity not seen with either agent alone. In contrast, with agents producing oxidative DNA damage repaired by bifunctional glycosylase-initiated BER, there is only weak sensitization by cotreatment with PARP inhibitor. Other clinically used DNA-damaging agents repaired by different DNA repair pathways also reveal minimal 4-AN-mediated sensitization. This information has potentially important implications for strategic use of PARP inhibitors in chemotherapy.

Regulation and function of DNA methylation in plants and animals

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review.

Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1

Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase IIIalpha-XRCC1 complex. Of these, ligase IIIalpha-XRCC1 is the most efficient. Moreover, ligase IIIalpha-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.

Oxidative stress alters base excision repair pathway and increases apoptotic response in apurinic/apyrimidinic endonuclease 1/redox factor-1 haploinsufficient mice

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-κB, and the major 5′-endonuclease in base excision repair (BER). We utilized mice containing a heterozygous gene-targeted deletion of APE1/Ref-1 ( Apex +/−) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-κB DNA-binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress, in which significant increases in GADD45g expression, p53 protein stability, and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UNG) and bifunctional (OGG1) DNA glycosylase-initiated BER in the liver of Apex +/− mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), whereas removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex +/− mice exposed to 2-NP displayed a significant decline in 3′-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA, suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase-initiated BER.

Coordinating the Initial Steps of Base Excision Repair: APURINIC/APYRIMIDINIC ENDONUCLEASE 1 ACTIVELY STIMULATES THYMINE DNA GLYCOSYLASE BY DISRUPTING THE PRODUCT COMPLEX

DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing apurinic/apyrimidinic (AP) DNA. For many glycosylases, the AP-DNA remains tightly bound, impeding enzymatic turnover. A prominent example is thymine DNA glycosylase (TDG), which removes T from G.T mispairs and recognizes other lesions, with specificity for damage at CpG dinucleotides. TDG turnover is very slow; its activity appears to reach a plateau as the [product]/[enzyme] ratio approaches unity. The follow-on base excision repair enzyme, AP endonuclease 1 (APE1), stimulates the turnover of TDG and other glycosylases, involving a mechanism that remains largely unknown. We examined the catalytic activity of human TDG (hTDG), alone and with human APE1 (hAPE1), using pre-steady-state kinetics and a coupled-enzyme (hTDG-hAPE1) fluorescence assay. hTDG turnover is exceedingly slow for G.T (k(cat)=0.00034 min(-1)) and G.U (k(cat)=0.005 min(-1)) substrates, much slower than k(max) from single turnover experiments, confirming that AP-DNA release is rate-limiting. We find unexpectedly large differences in k(cat) for G.T, G.U, and G.FU substrates, indicating the excised base remains trapped in the product complex by AP-DNA. hAPE1 increases hTDG turnover by 42- and 26-fold for G.T and G.U substrates, the first quantitative measure of the effect of hAPE1. hAPE1 stimulates hTDG by disrupting the product complex rather than merely depleting (endonucleolytically) the AP-DNA. The enhancement is greater for hTDG catalytic core (residues 111-308 of 410), indicating the N- and C-terminal domains are dispensable for stimulatory interactions with hAPE1. Potential mechanisms for hAPE1 disruption of the of hTDG product complex are discussed.

Substrate specificity and sequence-dependent activity of the Saccharomyces cerevisiae 3-methyladenine DNA glycosylase (Mag)

DNA glycosylases initiate base excision repair by first binding, then excising aberrant DNA bases. Saccharomyces cerevisiae encodes a 3-methyladenine (3MeA) DNA glycosylase, Mag, that recognizes 3MeA and various other DNA lesions including 1, N 6-ethenoadenine (ɛA), 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 ɛA and AP-sites the most tightly, followed by the cross-linked 1,2-d(ApG) cisplatin adduct. While ɛA 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 ɛA and Hx by Mag, when these lesions are present at different positions within A:T and G:C tracts. Overall, ɛA 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 ɛA relative to Hx, except in the TTXTT (X = ɛA 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.

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1(-/-) mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase beta (pol beta) is specific to this pathway, whereas pol beta is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1(-/-), and to a lesser extent in pol beta(-/-) cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol beta(-/-) cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1(-/-) cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

Steady‐state kinetics of human thymine DNA glycosylase (hTDG) using a coupled‐enzyme assay: Implications for the mechanism of hTDG stimulation by human AP endonuclease 1

DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing an abasic (AP) site in the DNA. Many glycosylases bind the AP DNA product tightly, impeding enzymatic turnover. Human thymine DNA glycosylase (hTDG), which recognizes G·T mispairs and other mutagenic lesions, exhibits severe product inhibition, precluding the use of steady-state kinetics to study its catalytic mechanism. To overcome this problem, we developed a coupled enzyme assay where the second enzyme, human AP endonuclease (hAPE1), stimulates the turnover of hTDG. The coupled reaction is monitored continuously by fluorescence spectroscopy using DNA containing 2-aminopurine adjacent to the target base. We determined the steady-state kinetic parameters for hTDG, and its catalytic core (hTDG-core, Phe111 to Val308) against a G·U substrate. We find that hAPE1 enhances the steady state turnover (kcat) of hTDG and hTDG-core by about 15-fold and 100-fold, respectively, and that hAPE1 actively displaces AP DNA from hTDG and hTDG-core. Thus, the 110 N-terminal residues and 102 C-terminal residues of hTDG are not required for its stimulation by hAPE1. Using the coupled assay, we show that hTDG-core is not inhibited by the substrate bases uracil or thymine at up to 5 mM concentrations. In contrast, hTDG-core is inhibited by duplex DNA containing a stable dU analog and by DNA containing a central CpG site.

Apurinic/Apyrimidinic Endonuclease (APE/REF-1) Haploinsufficient Mice Display Tissue-specific Differences in DNA Polymerase β-Dependent Base Excision Repair

Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5'-endonuclease activity and 3'-"end-trimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex(+/-)). Our data indicate that Apex(+/-) mice are indeed APE-haploinsufficient, as exhibited by a 40-50% reduction (p < 0.05) in APE mRNA, protein, and 5'-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase beta and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase beta-dependent BER response to cellular stress.

Long-patch DNA repair synthesis during base excision repair in mammalian cells.

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.

Hypersensitivity of DNA polymerase β null mouse fibroblasts reflects accumulation of cytotoxic repair intermediates from site-specific alkyl DNA lesions

Monofunctional alkylating agents react with DNA by S N 1 or S N 2 mechanisms resulting in formation of a wide spectrum of cytotoxic base adducts. DNA polymerase β (β-pol) is required for efficient base excision repair of N-alkyl adducts, and we make use of the hypersensitivity of β-pol null mouse fibroblasts to investigate such alkylating agents with a view towards understanding the DNA lesions responsible for the cellular phenotype. The inability of O 6-benzylguanine to sensitize wild-type or β-pol null cells to S N 1-type methylating agents indicates that the observed hypersensitivity is not due to differential repair of cytotoxic O-alkyl adducts. Using a 3-methyladenine-specific agent and an inhibitor of such methylation, we find that inefficient repair of 3-methyladenine is not the reason for the hypersensitivity of β-pol null cells to methylating agents, and further that 3-methyladenine is not the adduct primarily responsible for methyl methanesulfonate (MMS)- and methyl nitrosourea-induced cytotoxicity in wild-type cells. Relating the expected spectrum of DNA adducts and the relative sensitivity of cells to monofunctional alkylating agents, we propose that the hypersensitivity of β-pol null cells reflects accumulation of cytotoxic repair intermediates, such as the 5′-deoxyribose phosphate group, following removal of 7-alkylguanine from DNA. In support of this conclusion, β-pol null cells are also hypersensitive to the thymidine analog 5-hydroxymethyl-2′-deoxyuridine (hmdUrd). This agent is incorporated into cellular DNA and elicits cytotoxicity only when removed by glycosylase-initiated base excision repair. Consistent with the hypothesis that there is a common repair intermediate resulting in cytotoxicity following treatment with both types of agents, both MMS and hmdUrd-initiated cell death are preceded by a similar rapid concentration-dependent suppression of DNA synthesis and a later cell cycle arrest in G 0/G 1 and G 2M phases.

The Mammalian Mismatch Repair Pathway Removes DNA 8-oxodGMP Incorporated from the Oxidized dNTP Pool

Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSα and MutLα heterodimers [1]. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates [2]. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies [3]. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs [4]. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication [5]. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1—an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool—affords additional protection by minimizing 8-oxodGMP incorporation during replication [6]. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H2O2-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H2O2-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2−/− MEFs.

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice.

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.

Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover.

DNA glycosylases initiate base excision repair (BER) through the generation of potentially harmful abasic sites (AP sites) in DNA. Human thymine-DNA glycosylase (TDG) is a mismatch-specific uracil/thymine-DNA glycosylase with an implicated function in the restoration of G*C base pairs at sites of cytosine or 5-methylcytosine deamination. The rate-limiting step in the action of TDG in vitro is its dissociation from the product AP site, suggesting the existence of a specific enzyme release mechanism in vivo. We show here that TDG interacts with and is covalently modified by the ubiquitin-like proteins SUMO-1 and SUMO-2/3. SUMO conjugation dramatically reduces the DNA substrate and AP site binding affinity of TDG, and this is associated with a significant increase in enzymatic turnover in reactions with a G*U substrate and the loss of G*T processing activity. Sumoylation also potentiates the stimulatory effect of APE1 on TDG. These observations implicate a function of sumoylation in the controlled dissociation of TDG from the AP site and open up novel perspectives for the understanding of the molecular mechanisms coordinating the early steps of BER.

In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells.

3-Methyladenine (3MeA) DNA glycosylases initiate base excision repair by removing 3MeA. These glycosylases also remove a broad spectrum of spontaneous and environmentally induced base lesions in vitro. Mouse cells lacking the Aag 3MeA DNA glycosylase (also known as the Mpg, APNG or ANPG DNA glycosylase) are susceptible to 3MeA-induced S phase arrest, chromosome aberrations and apoptosis, but it is not known if Aag is solely responsible for repair of 3MeA in vivo. Here we show that in AAG:(-/-) cells, 3MeA lesions disappear from the genome slightly faster than would be expected by spontaneous depurination alone, suggesting that there may be residual repair of 3MeA. However, repair of 3MeA is at least 10 times slower in AAG:(-/-) cells than in AAG:(+/+) cells. Consequently, 24 h after exposure to [(3)H]MNU, 30% of the original 3MeA burden is intact in AAG:(-/-) cells, while 3MeA is undetectable in AAG:(+/+) cells. Thus, Aag is the major DNA glycosylase for 3MeA repair. We also investigated the in vivo repair kinetics of another Aag substrate, 7-methylguanine. Surprisingly, 7-methylguanine is removed equally efficiently in AAG:(+/+) and AAG:(-/-) cells, suggesting that another DNA glycosylase acts on lesions previously thought to be repaired by Aag.

Influence of DNA structure on hypoxanthine and 1,N(6)-ethenoadenine removal by murine 3-methyladenine DNA glycosylase.

3-Methyladenine DNA glycosylases initiate base excision repair by flipping the nucleotide bearing the target base out of double-stranded DNA into an active site pocket for glycosylic bond cleavage and base release. Substrate bases for the murine 3-methyladenine DNA glycosylase (other than 3-methyladenine) include hypoxanthine and 1,N(6)-ethenoadenine, two mutagenic adducts formed by both endogenous and exogenous agents. Using double-stranded DNA oligonucleotides containing damaged bases at specific sites, we studied the relative removal rates for these two adducts when located in different sequence contexts. One of the sequence contexts was an A:T tract, chosen because DNA secondary structure is known to change along the length of this tract, due to a progressive narrowing of the minor groove. Here we report that removal rates for hypoxanthine, but not for 1,N(6)-ethenoadenine, are dramatically affected by its location within the A:T tract. In addition, the removal rates of hypoxanthine and 1,N(6)-ethenoadenine when paired opposite thymine or cytosine were examined, and in each sequence context hypoxanthine removal decreased by at least 20-fold when paired opposite cytosine versus thymine. In contrast, 1, N(6)-ethenoadenine removal was unaffected by the identity of the opposing pyrimidine. We conclude that the removal of certain bases by the mouse 3-methyladenine DNA glycosylase can be modulated by both adjacent and opposing sequence contexts. The influence of DNA sequence context upon DNA repair rates, such as those described here, may contribute to the creation of mutational hot spots in mammalian cells.