Effects of chromium on DNA replication in vitro.

Chromium is an environmentally significant human carcinogen with complicated metabolism and an unknown mechanism of mutagenesis. Chromium(VI) is taken up by cells as the chromate anion and is reduced intracellularly via reactive intermediates to stable Cr(III) species. Chromium(III) forms tight complexes with biological ligands, such as DNA and proteins, which are slow to exchange. In vitro, CrCl3.6H2O primarily interacts with DNA to form outer shell charge complexes with the DNA phosphates. However, at micromolar concentrations, the Cr(III) binds to a low number of saturable tight binding sites on single-stranded M13 DNA. Additional chromium interacts in a nonspecific manner with the DNA and can form intermolecular DNA cross-links. Although high concentrations of Cr(III) inhibit DNA replication, micromolar concentrations of Cr(III) can substitute for Mg2+, weakly activate the Klenow fragment of E. coli DNA polymerase I (Pol I-KF), and act as an enhancer of nucleotide incorporation. Alterations in enzyme kinetics induced by Cr(III) increase DNA polymerase processivity and the rate of polymerase bypass of DNA lesions. This results in an increased rate of spontaneous mutagenesis during DNA replication both in vitro and in vivo. Our results indicate that chromium(III) may contribute to chromate-induced mutagenesis and may be a factor in the initiation of chromium carcinogenesis.


Introduction
Metal compounds, as a class, are among the best documented of all human carcinogens (1), but the mechanisms of metal carcinogenesis are not fully understood. Although it is often assumed that carcinogenesis is (at least in part) the result of DNA damage and mutagenesis, metals have many genotoxic effects and the relative importance of these different effects are not known. Each metal-and in many cases different compounds of a single metal-exhibits a unique spectrum of genotoxic effects. Cr(VI) or chromate, the biologically active form of environmental chromium, is taken up by cells and reduced intracellularly to reactive Cr(V) and Cr(IV) species and then to stable Cr(III). In the process, chromium induces oxidative DNA damage, DNA strand breaks, DNA-DNA and DNA-protein cross-links, and mutations (2)(3)(4). The genetic consequences of the different types of chromium complexes and the relative importance of oxidative This paper was presented at the Second International Meeting on Molecular Mechanisms of Metal Toxicity and Carcinogenicity held 10-17 January 1993 in Madonna di Campiglio, Italy.
The excellent technicial assistance of Li-Sha Xu is gratefully acknowledged. This research was supported in part by NIH  DNA damage are unknown. Although much of the published research on the mechanisms of chromium-induced genotoxicity has dealt with the complex intracellular metabolism of chromium and the DNA damage produced by Cr(VI) in whole cells (5,6), it is well established that only the reduced forms of chromium, such as Cr(V) and Cr(III), form complexes with DNA and proteins (6,7). Cr(III), for example, forms stable complexes with ligands on DNA, proteins, and small molecules such as glutathione. Since these complexes constitute the most persistent form of intracellular chromium their impact on chromium toxicology is of significance. The research outlined here describes the effects of Cr(III)-DNA complexes on DNA replication in vitro.

Materials and Methods
Ml 3mp2 single-stranded DNA was prepared as previously reported (8) using standard procedures (9). Unlabeled deoxynucleotides were obtained from Sigma Chemical Company (St. Louis, MO). 32p labeled deoxynucleoside triphosphates were from New England Nuclear (Boston, MA) and the M13 sequencing primers were either purchased from New England BioLabs (Beverly, MA) or synthesized by Dr. Bernard Goldschmidt, Department of Environmental Medicine, NYU Medical Center. Polymerase I-Klenow fragment was obtained from Pharmacia, Inc., Piscataway, NJ, and polynucleotide kinase was from New England BioLabs (Beverly, MA). All other reagents and chemicals were of molecular biology or DNA grade. Chromium solutions were made daily and all water was purified through a Milli-Q water system from Millipore.
Single-stranded M13mp2 DNA and Cr(Ill)-treated DNA was prepared and hybridized with a 2-fold excess of 17-mer primer ("-40 primers," New England BioLabs) as described previously (8). For measurement of nucleotide incorporation, the untreated and chromium-treated M13 DNA was primed and replicated in vitro using oX P-labeled dATP as a label and 0.02 to 0.2 units of DNA polymerase I-Klenow fragment (Pol I-KF). The reaction was stopped by the addition of disodium ethylenediamine tetraacetate (EDTA) and 10% trichloroacetic acid (TCA) (8).
Mutagenesis assays were performed as described previously (8) using an E. coli strain (NR9064) in which the mutS gene was inactivated by TN10 transduction giving a mismatch repair-deficient phenotype (10). SOS induction was achieved as described previously (11)  An oxidatively modified DNA template was prepared by incubating 5 pg of the single-stranded PCR products prepared as described above with 500 pM KMnO4 in a 25 pl final volume in the presence of 50 mM Tris-HCI, pH7.4, for 30 min at 37C. The oxidized DNA was purified by Sephadex G-50 spin columns and then treated with CrCI3.6H20 and hybridized with 5'-end labeled 15-mer primers (46-50 aa) as described above. The bypass of oxidative lesions (primarily thymine glycols) by Klenow fragment (0.01 units/reaction) was measured by primer-extension at 37°C for 5 min under the same conditions as for nucleotide incorporation. The extended primers were examined by 8% polyacrylamide sequencing gel electrophoresis. The dried gels were autoradiographed and the films were scanned by densitometer to quantify the bypass of lesions. 23% of the rate of Mg2+-catalyzed nucleotide incorporation measured in the same set of experiments. The mechanism of interaction between Cr(III) and the polymerase is not known. However, it is possible that the Cr(III) forms a bidentate-P,By-complex (12) with the dNTP at the dNTP binding site on the polymerase. This Cr(III)-complex may then facilitate the pyrophosphatase reaction by providing a better leaving group than a  The Cr(III)-mediated increase in polymerase processivity has been demonstrated by both primer extension analysis (8) and a newly developed competition assay using two different sized DNA templates (13). These assays show that Cr(III) bound to the DNA decreases the initiation of replication while, at the same time, the number of nucleotides incorporated per initiated template (a measure of processivity) is increased from 200% to 470% (8,13). The results suggest that the rate of binding of the polymerase to the Cr(III)-treated template is decreased, but that once bound the polymerase can incorporate more nucleotides before dissociating from the template.

Results and Discussion
The effects of Cr(III) on DNA polymerase processivity are not limited to Pol I-KF; other DNA polymerases show increased rates of nucleotide incorporation and increased processivity in the presence of Cr(III). We have shown, for example, that calf thymus polymerase a and cloned human DNA polymerase m both show enhanced processivity in the presence of Cr(III) (14,15). The most dramatic effect observed during replication of singlestranded M13mp2 DNA by polymerase a in the presence of CrCl3 is the loss of normal polymerase pause sites (16). Although polymerase I is not very active on a single stranded DNA template when Mg2+ is used as the cofactor; both Mn(II) (17) and Cr(III) (data not shown) are able to increase the activity of pol 0 on singlestranded M13 DNA (14,16). Not all polymerases are activated by Cr(III), however. T4 phage DNA polymerase, a normally processive enzyme, is inhibited by as little as 0.5 pM Cr(III) and does not show any significant activation by this trivalent cation, even in the absence of Mg (data not shown).

Mutagenic Effets ofCr(IU)
The effect of Cr(III) on the fidelity of eukaryotic enzymes is unknown; however, we have shown that the presence of Cr(III) during replication by E. coli DNA polymerases either in vitro or in vivo is mutagenic. A 2-to 4-fold increase in mutagenesis by Cr(III) was previously reported using the lacZa gene of Ml3mp2 as a mutagenic target (8,14,15). More recently we have found that, if a mutant M13 phage DNA (Ml3mp2-C141) is replicated in the presence of Cr(III) in a mismatch repair deficient E. coli host, reversion mutagenesis is increased up to 94-fold in non-SOS-induced cells, but not in SOS-induced cells ( Table 1). The chromium-dependent increase in mutagenesis requires the use of freshly treated DNA. If the chromium is allowed to equilibrate between the DNA and the buffer by storing the treated DNA (from which the unbound Cr(III) was removed by gel filtration) for 23 days before transfection no mutagenesis is seen. Some residual damage or bound chromium in the 10 pM treated sample does, however, produce a 46% decrease in phage recovery (Table 1).
In contrast to the strong mutagenic response seen in mismatch repair-deficient cells, reversion mutagenesis by Cr(III) only reaches a maximum of 2-to 3-fold above background in a mismatch repair-proficient host (8). This suggests that most of the errors introduced during replication in the presence of Cr(III) can be repaired by either mismatch repair or are mitigated by SOS-associated processes. This may indicate that the RecA protein, a single-strand binding protein present at high concentration in the SOS-induced cells, can displace the Cr(III) from the DNA and thus mitigate the effects of the metal ion on replication. It can also be seen in Table 1 that 10 pM Cr(III) [a concentration which induces significant amounts of DNA crosslinking (8)] is significantly more mutagenic than 5 pM Cr(III) under non-SOS conditions, but not after SOS-induction. This implies that base substitution mutagenesis by Cr(III) may be promoted by DNA crosslinking and that the resulting mismatches may be repaired by either the mismatch repair system or by SOS-dependent processing.
Several of the Cr(III)-induced and spontaneous mutants were isolated and sequenced ( Table 2). The results show that Cr(III) produces a significant increase in the frequency of C-+A transversion mutations (at C141) relative to the spontaneous mutant spectrum. DNA replication in the presence of Cr(III) produced 31% C->A transversions, 19% C-+G transversions, and 50% C-*T transitions at target sites C141 and C142; whereas the spontaneous mutations at the same sites consisted of 9% C-+A transversions; 27% C-*G transversions, and 64% C->T transitions. Spontaneous mutagenesis in a mismatch repair-deficient host has been shown to consist primarily of an increased frequency of transition mutations (10); therefore, this increase in transversions induced by Cr(III) may indicate a significant alteration in the type of misincorporation that occurs during DNA replication in the presence of this metal ion.

Cr(Il) Increases Polymerase Bypass of DNA Damage
The evidence for increased polymerase processivity, along with increased mutagenesis in the presence of Cr(III), led us to ask whether Cr(III) can increase polymerase bypass of other types of DNA damage. Since it has been shown that the intracellular reduction of chromium(VI) produces oxidative base damage (17,18), we investigated the ability of Cr(III) to promote bypass of oxidative DNA lesions. A 167nucleotide single-stranded fragment of Ml3mp2 DNA was prepared and oxidized with KMnO4, producing enough DNA damage (mostly thymidine glycols) to almost completely block replication at or before the first template thymidine (19). The KMnO4-treated DNA was further treated with 0 to 25 pM CrCl3 and then primed with an y32P-labeled primer.
Replication of the treated primer-template was carried out with 0.01 unit of Pol I-KF and the products of replication were analyzed on an 8% polyacrylamide/7 M urea sequencing gel. In the absence of Cr(III), replication was limited and barely reached the first putative thymidine glycol ( Figure  1). Fewer than 1% of the primers were extended beyond the first template thymine residue. After treatment of the oxidized DNA with 1 to 5 pM Cr(III), replication was greatly extended and increased bypass of the oxidized DNA base The number of C-+A, C ->G, and C-*T mutations at each of the target sites is given for a total of 11 spontaneous and 16 Cr-induced mutants.
Volume 102, Supplement 3, September 1994 damage was evident ( Figure 1) (13). In the presence of up to 5 pM Cr(III), from 5% to 29% of the primers were extended beyond the first thymidine. Higher concentrations of Cr(III), in the range of 10 to 25 pM, led to the formation of DNA-DNA crosslinks (seen at the top of the gel, to the right of the figure) and decreased polymerase activity. We are currently investigating the fidelity and kinetics of nucleotide incorporation opposite the oxidative lesions to understand the mechanism by which Cr(III) mediates the bypass of polymerase blocking lesions. In summary, micromolar concentrations of Cr(III) bound to the DNA template can increase DNA polymerase processivity and decrease DNA replication fidelity. These alterations in DNA function can result in greatly increased bypass of oxidative DNA lesions. Since several oxidative base lesions, such as 8oxodeoxyguanine have been shown to be promutagenic (20), Cr(III) may serve to increase the mutagenic potential of these lesions and contribute to the genotoxic effects of chromium in vivo.