Inhibition of the puromycin reaction with benzamidine and related compounds

Incubation of mouse cells with N-methyl-N′-nitro-N-nitrosoguanidine causes a strong inhibition of DNA replication the extent of which varies with the cell line used. Analysis of the products synthesized in drug-treated cells indicates a particularly severe effect on the joining of replicons while other steps in DNA synthesis like initiation and chain elongation are much less affected. The data indicate that replicon fusion may be extremely sensitive to changes in the topology of DNA induced by the introduction of rare single-strand breaks during repair of N-methylated purines produced by incubation of cells with small amounts of the methylating agent     

Poly(ADP-ribose) polymerase in base excision repair: always engaged,but not essential for DNA damage processing

Poly(ADP-ribose) polymerase (PARP-1) is an abundant nuclear protein with a high affinity for single- and double-strand DNA breaks. Its binding to strand breaks promotes catalysis of the covalent modification of nuclear proteins with poly(ADP-ribose) synthesised from NAD(+). PARP-1-knockout cells are extremely sensitive to alkylating agents, suggesting the involvement of PARP-1 in base excision repair; however, its role remains unclear. We investigated the dependence of base excision repair pathways on PARP-1 and NAD(+) using whole cell extracts derived from normal and PARP-1 deficient mouse cells and DNA substrates containing abasic sites. In normal extracts the rate of repair was highly dependent on NAD(+). We found that in the absence of NAD(+) repair was slowed down 4-6-fold after incision of the abasic site. We also established that in extracts from PARP-1 deficient mouse cells, repair of both regular and reduced abasic sites was increased with respect to normal extracts and was NAD(+)-independent, suggesting that in both short- and long-patch BER PARP-1 slows down, rather than stimulates, the repair reaction. Our data support the proposal that PARP-1 does not play a major role in catalysis of DNA damage processing via either base excision repair pathway.     

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O⁶-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N′-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced γH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.     

Delocalization of nucleolar poly(ADP-ribose) polymerase-1 to the nucleoplasm and its novel link to cellular sensitivity to DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by binding to DNA breaks, which causes PARP-1 automodification. PARP-1 activation is required for regulating various cellular processes, including DNA repair and cell death induction. PARP-1 involved in these regulations is localized in the nucleoplasm, but approximately 40% of PARP-1 can be found in the nucleolus. Previously, we have reported that nucleolar PARP-1 is delocalized to the nucleoplasm in cells exposed to DNA-damaging agents. However, the functional roles of this delocalization in cellular response to DNA damage is not well understood, since this approach simultaneously induces the delocalization of PARP-1 and its automodification. We therefore devised an approach for separating these processes. Unmodified PARP-1 was first delocalized from the nucleolus using camptothecin. Then, PARP-1 was activated by exposure of cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In contrast to treatment with MNNG alone, delocalization of PARP-1 by CPT, prior to its activation by MNNG, induced extensive automodification of PARP-1. DNA repair activity and consumption of intracellular NAD⁺ were not affected by this activation. On the other hand, activation led to an increased formation of apoptotic cells, and this effect was suppressed by inhibition of PARP-1 activity. These results suggest that delocalization of PARP-1 from the nucleolus to the nucleoplasm sensitizes cells to DNA damage-induced apoptosis. As it has been suggested that the nucleolus has a role in stress sensing, nucleolar PARP-1 could participate in a process involved in nucleolus-mediated stress sensing.     

p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

The cell cycle inhibitor p21^CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.     

DNA repair and chromosomal stability in the alkylating agent-hypersensitive Chinese hamster cell line 27-1

27-1 is a mutant of Chinese hamster ovary cells (CHO cells) that is hypersenstivie to the toxic effects of ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and other monofunctional alkylating agents. We show here that the enhanced MNNG sensitivity of these cells is not due to alterations in the amount of DNA methylation products introduced nor by a defect in the first step of removal of the main alkylation products 7-methylguanine and 3-methyladenine. However, these mutant cells perform more DNA repair synthesis after treatment with MNNG than normal CHO-9 cells. This observation might indicate a possible defect of a ligase involved in sealing DNA repair patches.27-1 cells did not show elevated frequencies of sister-chromatid exchange and chromosomal aberration induced by MNNG. The data show that MNNG-induced cell killing is not necessarily related to increased chromosomal instability.     

Response of human keratinocytes to extremely low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine

Since alkylating agents are widely present in the environment and constitute a continuous challenge to genome integrity, cells and organisms have developed defense mechanisms to remove such lesions. We monitored the response of human keratinocytes to a very low concentration of a methylating agent, namely 2.5 nM N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The effect of a 60-min exposure of quiescent cells to 2.5 nM MNNG was studied in terms of DNA integrity, poly(ADP-ribose) metabolism, clonogenic survival and DNA synthesis. We observed two waves of DNA strand break formation and resealing. Interestingly, the amount of DNA strand breaks in exposed cells was lower than in unexposed control cells. This phenomenon was also observed when cells were exposed to MNNG in the presence of a protein synthesis inhibitor, or when they were maintained on ice during the treatment. A dose of 2.5 nM MNNG stimulated poly(ADP-ribose) turnover, reduced the intracellular NAD⁺ content, stimulated DNA synthesis and caused a remarkable increase in clonogenic survival. Thus, the cellular responses to extremely low concentrations of MNNG differ sharply from those observed at higher doses of this carcinogen. We conclude that the very low dose response cannot be extrapolated from usual dose-response analyses.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.     

Vertebrate cells lacking FEN-1 endonuclease are viable but hypersensitive to methylating agents and H2O2

The structure-specific FEN-1 endonuclease has been implicated in various cellular processes, including DNA replication, repair and recombination. In vertebrate cells, however, no in vivo evidence has been provided so far. Here, we knocked out the FEN-1 gene (FEN1) in the chicken DT40 cell line. Surprisingly, homozygous mutant (FEN1^–/–) cells were viable, indicating that FEN-1 is not essential for cell proliferation and thus for Okazaki fragment processing during DNA replication. However, compared with wild-type cells, FEN1^–/– cells exhibited a slow growth phenotype, probably due to a high rate of cell death. The mutant cells were hypersensitive to methylmethane sulfonate, N-methyl-N′-nitro-N-nitrosoguanidine and H₂O₂, but not to UV light, X-rays and etoposide, suggesting that FEN-1 functions in base excision repair in vertebrate cells.     

The Escherichia coli alkylation response protein AidB is a redox partner of flavodoxin and binds RNA and acyl carrier protein

Microorganisms are exposed to a wide variety of exogenous and endogenous chemical agents that alkylate DNA. Escherichia coli cells exhibit an adaptive response that recognizes and repairs alkylated DNA lesions using Ada, AlkA, and AlkB enzymes. Another alkylation response protein, the DNA-binding flavoprotein AidB, was proposed to repair DNA or protect it from chemical alkylating agents, but direct evidence for its role is lacking. Here, AidB was shown to form tight complexes with both flavodoxin and acyl carrier protein. In addition, electron transfer between 1-electron and 2-electron reduced flavodoxin to oxidized AidB was observed, although with very small rate constants. AidB was found to bind to RNA, raising the prospect that the protein may have a role in protection of RNA from chemical alkylation. Finally, the reagent N-methyl-N′-nitro-N-nitrosoguanidine was eliminated as a direct substrate of the enzyme.     

Hydrazine and methylhydrazine as recA+-independent mutagens in Escherichia coli

The ultimate cause of both physical and chemical mutagenesis is assumed in most cases, to be some inducible error-prone repair process acting on the lesions caused by the mutagenic agent [7]. In Escherichia coli this mutagenic pathway seems to be dependent on both recA⁺ and lexA⁺ loci [10]. There are some mutagens, however, such as simple base analogs or some strong alkylating agents, such as N-methyl-N′-nitro-N′-nitrosoguanidine, that probably act by simply causing mis-pairing of bases in DNA. There is some evidence, based on work done with bacteriophages [2] or with Haemophilus influenzae [6], that hydrazine also acts by producing errors in base pairing. In this paper, further evidence is reported in support of this theory by showing that the recA lexA genotype does not affect the mutagenic properties of either hydrazine or methylhydrazine in Escherichia coli.     

Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1

The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo- and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase beta, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.     

Protective effect of nicotinamide against poly(ADP-ribose) polymerase-1-mediated astrocyte death depends on its transporter-mediated uptake

AimPoly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair enzyme, and its excessive activation, following ischemia, trauma, etc., depletes cellular nicotinamide adenine dinucleotide (NAD⁺) as a substrate and eventually leads to brain cell death. Nicotinamide, an NAD⁺ precursor and a PARP-1 inhibitor, is known to prevent PARP-1-triggered cell death, but there is no available information on the mechanisms involved in its transport. Here we clarified the transport characteristics of nicotinamide in primary cultured mouse astrocytes.Main methodsUptake characteristics of [¹⁴C]nicotinamide were assessed by a conventional method with primary cultured mouse astrocytes. Cell viability and PARP-1 activity were determined with intracellular LDH activity and immunocytochemical detection of PAR accumulation, respectively.Key findingsPARP-1 activation was induced by treatment of astrocytes with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent. MNNG-triggered astrocyte death and PAR accumulation were completely inhibited by treatment with nicotinamide as with DPQ (3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone), a second generation PARP inhibitor. The uptake of [¹⁴C]nicotinamide was time-, temperature-, concentration- and pH-dependent, and was inhibited and stimulated by co- and pre-treatment with N-methylnicotinamide, a representative substrate of an organic cation transport system, respectively. Co-treatment of astrocytes with nicotinamide and N-methylnicotinamide resulted in a decrease in PAR accumulation and absolute prevention of cell death.SignificanceThese findings suggest that nicotinamide has a protective effect against PARP-1-induced astrocyte death and that its transporter-mediated uptake, which is extracellular pH-sensitive and common to N-methylnicotinamide, is critical for prevention of PARP-1-triggered cell death.     

Poly(ADP-ribose) polymerase-1 modulates DNA repair capacity and prevents formation of DNA double strand breaks

Although poly(ADP-ribose) polymerase-1 (PARP-1) has no enzymatic activity involved in DNA damage processing by the base excision repair (BER) pathway, PARP-1 deficient cells are genetically unstable and sensitive to DNA-damaging agents. To explain this paradox, we investigated the impact of PARP-1 on BER in mammalian cells. We reduced cellular PARP-1 protein levels using siRNA, then introduced DNA damage by hydrogen peroxide treatment and examined the repair response. We find that PARP-1 is not involved in recruitment of the major BER proteins to sites of DNA damage. However, we find that PARP-1 protects excessive DNA single strand breaks (SSBs) from converting into DNA double strand breaks (DSBs) thus preserving them for subsequent repair by BER enzymes. This suggests that PARP-1 plays an important role in BER by extending the ability of BER enzymes to process DNA single strand breaks arising directly after mutagen stress or during processing of DNA lesions following extensive DNA damage.     

The role of DNA polymerases in DNA repair in Escherichia coli: DNA polymerase I-dependent repair following chemical alkylation of permeabilized bacteria.

Many mutagens and carcinogens damage DNA and elicit repair synthesis in cells. In the present study we report that alkylation of the DNA of Escherichia coli that have been made permeable to nucleotides by toluene treatment results in the expression of a DNA polymerase I-directed repair synthesis. The advantage of the system described here is that it permits measurement of only DNA polymerase I-directed repair synthesis and serves as a simple, rapid method for determining the ability of a given chemical to elicit “excision-repair” in bacteria.DNA ligation is intentionally prevented in our system by addition of the inhibitor nicotinamide mononucleotide. In the absence of DNA ligase activity, nick translation is extensive and an “exaggerated” repair synthesis occurs. This amplification of repair synthesis is unique for DNA polymerase I since it is not observed in mutant cells deficient in this polymerase. DNA ligase apparently controls the extent of nucleotide replacement by this repair enzyme through its ability to rejoin “nicks” thereby terminating the DNA elongation process.The nitrosoamides N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea, as well as the nitrosoamidines N-methyl-N′-nitro-N-nitrosoguanidine and N-ethyl-N′-nitro-N-nitrosoguanidine, elicit DNA polymerase I-directed repair synthesis. Methyl methanesulphonate is especially potent in this regard, while its ethyl derivative, ethyl methanesulphonate, is a poor inducer of DNA polymerase I activity in permeabilized cells.     

Correlation between lethality and DNA single-strand breaks in Bacillus subtilis cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

The effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment two Bacillus subtilis strains which exhibit different UV sensitivities were monitored at both the cellular (survival) and subcellular (DNA strand break) levels. The MNNG-induced single strand DNA breaks (SSB) in either strain, as measured by alkaline sucrose gradient centrifugation, were shown to be well correlated with lethality. These DNA lesions were shown by a computer simulation to be randomly induced. Cell survival after MNNG treatment is inversely related to the number of replication forks per cell which in turn depends upon the doubling time of the culture and the growth phase. The production of single-strand breaks and cell killing are proportional to the log of the initial MNNG concentration and may imply that decomposition of the mutagen is (pseudo) second order.     

The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase

Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9–Rad1–Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67–110 of hTDG, and Val74 of hTDG plays an important role in the TDG–Hus1 interaction. In contrast to the core domain of hTDG (residues 110–308), hTDG(67–308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER.     

Antimutagenic effect of lectin and <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="Italic">N’</Emphasis>-nitro-<Emphasis Type="Italic">N</Emphasis>-nitrosoguanidine on induced mutagenesis in mammalian cells in vitro

Factors and ways in which macromolecules influence the mutation process are considered. An antimutagenic effect is demonstrated in a study of the combined influence of lectins and the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) on mutagenesis in Chinese hamster cells. Thus, in different schemes of treatment of cells with albumin and MNNG, the experimental frequency of mutations induced by the two agents was statistically reliably lower than the theoretically expected level for their independent combined action. The possibility that there exist common targets and mechanisms through which different types of mutagenic agents act on the cell DNA is discussed.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.