Inhibition of Ape1 Nuclease Activity by Lead, Iron, and Cadmium
Inhibition of Ape1 Nuclease Activity by Lead, Iron, and Cadmium
Many environmental metals are co-carcinogens, eliciting their effects via inhibition of DNA repair. Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is the major mammalian abasic endonuclease and initiates repair of this cytotoxic/mutagenic lesion by incising the DNA backbone via a Mg-dependent reaction. In this study we examined the effects of arsenite [As(III)], cadmium [Cd(II)], cobalt [Co(II)], iron [Fe(II)], nickel [Ni(II)], and lead [Pb(II)] at concentrations ranging from 0.3 to 100 µM on the incision activity of Ape1 in the presence of 1 mM MgCl2. Pb(II) and Fe(II) inhibited Ape1 activity at each of the concentrations tested, with an IC50 (half-maximal inhibitory concentration) of 0.61 and 1.0 µM, respectively. Cd(II) also inhibited Ape1 activity but only at concentrations > 10 µM. No inhibition was seen with As(III), Co(II), or Ni(II). A similar inhibition pattern was observed with the homologous Escherichia coli protein, exonuclease III, but no inhibition was seen with the structurally distinct AP endonuclease E. coli endonuclease IV, indicating a targeted effect of Pb(II), Fe(II), and Cd(II) on the Ape1-like repair enzymes. Excess nonspecific DNA did not abrogate the metal inactivation, suggesting a protein-specific effect. Notably, Cd(II), Fe(II), and Pb(II) [but not As(III), Co(II), or Ni(II)] inhibited AP endonuclease activity in whole-cell extracts but had no significant effect on single nucleotide gap filling, 5´-flap endonuclease, and nick ligation activities, supporting the idea of selective inactivation of Ape1 in cells. Our results are the first to identify a potential DNA repair enzyme target for lead and suggest a means by which these prevalent environmental metals may elicit their deleterious effects.
Toxic metal compounds are widely distributed in the environment and are frequently used in industrial processes (Hayes 1997). Because of their extended persistence in biological systems and their tendency to accumulate in certain tissues, they represent important environmental and occupational hazards. Epidemiologic studies and animal trials have shown many metal compounds to be carcinogenic, although they elicit only mild mutagenic effects in bacterial test systems or in mammalian cell culture (Hartwig et al. 2002; Hartwig and Schwerdtle 2002).
DNA repair systems function to correct DNA damage that arises spontaneously or due to exposure to certain environmental agents (Hoeijmakers 2001). It is well documented that reduced DNA repair capacity can lead to genetic instability and thus human disease, most notably cancer. Because cell biology studies indicate that many metal compounds enhance the genotoxic effects of known mutagens such as ultraviolet C radiation, X rays, benzo[a]pyrene, cisplatin, and DNA alkylating agents, it has been postulated that environmental metals are comutagenic and potentiate the carcinogenic effects of DNA-damaging agents by inhibiting DNA repair processes (Hartwig et al. 2002; Hartwig and Schwerdtle 2002; Hayes 1997). Consistent with this notion, both in vitro and in vivo studies have shown that certain metal compounds can inhibit specific DNA repair events.
For example, Cd(II), Cu(II), Co(II), and Ni(II) inhibit (at concentrations ≥ 200 µM) the in vitro DNA-binding activity of the human xeroderma pigmentosum group A protein, a critical damage recognition factor in nucleotide excision repair (Asmuss et al. 2000). Poly(adenosine diphosphate-ribose)polymerase 1, which operates as a molecular sensor in DNA strand-break responses, is inactivated by Ni(II), Co(II), Cd(II), Cu(II), and very low concentrations (10 nM) of As(III) (Hartwig et al. 2003). The DNA-binding capacity of the tumor suppressor protein p53 is impaired by Cd(II), Ni(II), and Co(II) (Meplan et al. 1999; Palecek et al. 1999). In addition, Cd(II) and Zn(II) (at ≥ 100 µM) inactivate the in vitro DNA glycosylase activity of Ogg1, a mammalian repair enzyme that functions to excise damaged/mutagenic bases, such as 8-oxoguanine, from DNA (Zharkov and Rosenquist 2002).
Cd(II) was recently shown to inhibit DNA mismatch repair (MMR) (Jin et al. 2003), a process known to correct replication errors; deficiencies in this pathway have been linked to the development of hereditary nonpolyposis colorectal cancer (Heinen et al. 2002). Although the precise target for MMR inhibition remains unclear, Jin et al. (2003) found that yeast chronically exposed to environmental concentrations of Cd(II) display an increased mutation rate genetically dependent on an intact MMR pathway. Thus, studies are beginning to unveil the molecular targets of environmental metals and the mechanisms by which they may elicit their co-mutagenic effects and, hence, carcinogenic potential.
Apurinic/apyrimidinic (AP) sites are frequent lesions in DNA, generated by spontaneous, damage-induced, or enzyme-catalyzed hydrolysis of the N-glycosylic bond, which attaches the base moiety to the sugar residue (Wilson and Barsky 2001). If unrepaired, these noncoding lesions present both cytoxic and mutagenic challenges to the cell. AP endonuclease 1 (Ape1) is the major mammalian abasic endonuclease, accounting for > 95% of the total cellular AP site incision activity (Demple and Harrison 1994). This enzyme initiates repair of AP sites by cleaving the phosphodiester backbone 5´ to the damage site, a critical step in the base excision repair (BER) pathway, which handles most spontaneous, alkylation, and oxidative DNA damage (Kelley et al. 2003). In addition to its role as an AP endonuclease, Ape1 functions in specific strand-break contexts to excise 3´-oxidative blocking termini (e.g., phosphoglycolate and phosphate damages) (Suh et al. 1997; Wilson 2003; Winters et al. 1994), as well as certain 3´-mismatched nucleotides (Chou and Cheng 2002; Hadi et al. 2002), via its 3´- to 5´-phosphodiesterase/exonuclease activity. Each of these Ape1 repair functions is carried out by the same metal (magnesium)-dependent catalytic reaction (Gorman et al. 1997; Mol et al. 2000b).
Animal studies have shown that heterozygous (reduced-function) APE1 mice, which maintain 50% Ape1 repair activity, exhibit increased risk for phenotypic consequences associated with oxidative stress, most notably reduced survival and elevated cancer susceptibility (Meira et al. 2001). Because both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy studies revealed that lead can bind the active site of Ape1 (Beernink et al. 2001; Lowry et al. 2003), we explored the effects of several heavy metals, at presumed biological concentrations, on Ape1 DNA repair function. In this article, we demonstrate that Pb(II) and Fe(II), and to a lesser degree Cd(II), inhibit Ape1 nuclease activity, unveiling a novel molecular target for these environmental metals.
Many environmental metals are co-carcinogens, eliciting their effects via inhibition of DNA repair. Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is the major mammalian abasic endonuclease and initiates repair of this cytotoxic/mutagenic lesion by incising the DNA backbone via a Mg-dependent reaction. In this study we examined the effects of arsenite [As(III)], cadmium [Cd(II)], cobalt [Co(II)], iron [Fe(II)], nickel [Ni(II)], and lead [Pb(II)] at concentrations ranging from 0.3 to 100 µM on the incision activity of Ape1 in the presence of 1 mM MgCl2. Pb(II) and Fe(II) inhibited Ape1 activity at each of the concentrations tested, with an IC50 (half-maximal inhibitory concentration) of 0.61 and 1.0 µM, respectively. Cd(II) also inhibited Ape1 activity but only at concentrations > 10 µM. No inhibition was seen with As(III), Co(II), or Ni(II). A similar inhibition pattern was observed with the homologous Escherichia coli protein, exonuclease III, but no inhibition was seen with the structurally distinct AP endonuclease E. coli endonuclease IV, indicating a targeted effect of Pb(II), Fe(II), and Cd(II) on the Ape1-like repair enzymes. Excess nonspecific DNA did not abrogate the metal inactivation, suggesting a protein-specific effect. Notably, Cd(II), Fe(II), and Pb(II) [but not As(III), Co(II), or Ni(II)] inhibited AP endonuclease activity in whole-cell extracts but had no significant effect on single nucleotide gap filling, 5´-flap endonuclease, and nick ligation activities, supporting the idea of selective inactivation of Ape1 in cells. Our results are the first to identify a potential DNA repair enzyme target for lead and suggest a means by which these prevalent environmental metals may elicit their deleterious effects.
Toxic metal compounds are widely distributed in the environment and are frequently used in industrial processes (Hayes 1997). Because of their extended persistence in biological systems and their tendency to accumulate in certain tissues, they represent important environmental and occupational hazards. Epidemiologic studies and animal trials have shown many metal compounds to be carcinogenic, although they elicit only mild mutagenic effects in bacterial test systems or in mammalian cell culture (Hartwig et al. 2002; Hartwig and Schwerdtle 2002).
DNA repair systems function to correct DNA damage that arises spontaneously or due to exposure to certain environmental agents (Hoeijmakers 2001). It is well documented that reduced DNA repair capacity can lead to genetic instability and thus human disease, most notably cancer. Because cell biology studies indicate that many metal compounds enhance the genotoxic effects of known mutagens such as ultraviolet C radiation, X rays, benzo[a]pyrene, cisplatin, and DNA alkylating agents, it has been postulated that environmental metals are comutagenic and potentiate the carcinogenic effects of DNA-damaging agents by inhibiting DNA repair processes (Hartwig et al. 2002; Hartwig and Schwerdtle 2002; Hayes 1997). Consistent with this notion, both in vitro and in vivo studies have shown that certain metal compounds can inhibit specific DNA repair events.
For example, Cd(II), Cu(II), Co(II), and Ni(II) inhibit (at concentrations ≥ 200 µM) the in vitro DNA-binding activity of the human xeroderma pigmentosum group A protein, a critical damage recognition factor in nucleotide excision repair (Asmuss et al. 2000). Poly(adenosine diphosphate-ribose)polymerase 1, which operates as a molecular sensor in DNA strand-break responses, is inactivated by Ni(II), Co(II), Cd(II), Cu(II), and very low concentrations (10 nM) of As(III) (Hartwig et al. 2003). The DNA-binding capacity of the tumor suppressor protein p53 is impaired by Cd(II), Ni(II), and Co(II) (Meplan et al. 1999; Palecek et al. 1999). In addition, Cd(II) and Zn(II) (at ≥ 100 µM) inactivate the in vitro DNA glycosylase activity of Ogg1, a mammalian repair enzyme that functions to excise damaged/mutagenic bases, such as 8-oxoguanine, from DNA (Zharkov and Rosenquist 2002).
Cd(II) was recently shown to inhibit DNA mismatch repair (MMR) (Jin et al. 2003), a process known to correct replication errors; deficiencies in this pathway have been linked to the development of hereditary nonpolyposis colorectal cancer (Heinen et al. 2002). Although the precise target for MMR inhibition remains unclear, Jin et al. (2003) found that yeast chronically exposed to environmental concentrations of Cd(II) display an increased mutation rate genetically dependent on an intact MMR pathway. Thus, studies are beginning to unveil the molecular targets of environmental metals and the mechanisms by which they may elicit their co-mutagenic effects and, hence, carcinogenic potential.
Apurinic/apyrimidinic (AP) sites are frequent lesions in DNA, generated by spontaneous, damage-induced, or enzyme-catalyzed hydrolysis of the N-glycosylic bond, which attaches the base moiety to the sugar residue (Wilson and Barsky 2001). If unrepaired, these noncoding lesions present both cytoxic and mutagenic challenges to the cell. AP endonuclease 1 (Ape1) is the major mammalian abasic endonuclease, accounting for > 95% of the total cellular AP site incision activity (Demple and Harrison 1994). This enzyme initiates repair of AP sites by cleaving the phosphodiester backbone 5´ to the damage site, a critical step in the base excision repair (BER) pathway, which handles most spontaneous, alkylation, and oxidative DNA damage (Kelley et al. 2003). In addition to its role as an AP endonuclease, Ape1 functions in specific strand-break contexts to excise 3´-oxidative blocking termini (e.g., phosphoglycolate and phosphate damages) (Suh et al. 1997; Wilson 2003; Winters et al. 1994), as well as certain 3´-mismatched nucleotides (Chou and Cheng 2002; Hadi et al. 2002), via its 3´- to 5´-phosphodiesterase/exonuclease activity. Each of these Ape1 repair functions is carried out by the same metal (magnesium)-dependent catalytic reaction (Gorman et al. 1997; Mol et al. 2000b).
Animal studies have shown that heterozygous (reduced-function) APE1 mice, which maintain 50% Ape1 repair activity, exhibit increased risk for phenotypic consequences associated with oxidative stress, most notably reduced survival and elevated cancer susceptibility (Meira et al. 2001). Because both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy studies revealed that lead can bind the active site of Ape1 (Beernink et al. 2001; Lowry et al. 2003), we explored the effects of several heavy metals, at presumed biological concentrations, on Ape1 DNA repair function. In this article, we demonstrate that Pb(II) and Fe(II), and to a lesser degree Cd(II), inhibit Ape1 nuclease activity, unveiling a novel molecular target for these environmental metals.
