Sequence specificity of Cr(III)–DNA adduct formation in the p53 gene: NGG sequences are preferential adduct-forming sites

Abstract

Hexavalent chromium [Cr(VI)] is a known etiological factor in human lung cancer. Cr(VI) exposure-related lung cancer has a high mutation incidence in the p53 gene. Upon intake in human cells Cr(VI) is reduced to Cr(III), which is able to conjugate with amino acids and consequently form either binary Cr(III)-DNA or ternary Cr(III)-amino acid-DNA adducts. Both binary and ternary Cr(III)-DNA adducts are mutagenic. We have found that the Escherichia coli nucleotide excision enzyme UvrABC nuclease is able to incise Cr(III)- and Cr(III)-histidine-modified plasmid DNA and the extent of incision is proportional to the amount of Cr(III)-DNA adducts in the plasmid. In order to determine the role of Cr(III)-DNA adducts in the mutagenesis of the p53 gene in human cancer using the UvrABC nuclease incision method, we have mapped the Cr(III)-DNA distribution in PCR DNA fragments amplified from exons 5, 7 and 8 of the p53 gene. We have found that the sequence specificities of Cr(III)-DNA and Cr(III)-histidine-DNA adducts in the p53 gene sequence are identical and that both types of adducts are preferentially formed at -NGG- sequences, including codons 245, 248 and 249, the mutational hotspots in human lung cancer. It has been found that Cr(III)-DNA adducts induce mainly G to T mutations. Therefore, these results suggest that Cr(III)-DNA adduct formation contributes to the p53 gene mutations in lung carcinogenesis.

Polymerase η suppresses telomere defects induced by DNA damaging agents

“…Hexavalent chromium (Cr(VI)) is another environmental genotoxic agent that induces a spectrum of adducts including bulky lesions that are repaired by NER (25). Evidence indicates that Cr(VI) preferentially reacts with guanine runs (26), which predicts that telomeres are also susceptible to Cr(VI)-induced lesions. Consistent with this, we previously reported that Cr(VI)-induced replication stress causes telomere loss and aberrations (27).…”

Recognition and Incision of Cr(III) Ligand-Conjugated DNA Adducts by the Nucleotide Excision Repair Proteins UvrABC: Importance of the Cr(III)−Purine Moiety in the Enzymatic Reaction

“…Furthermore, we have found that UvrABC incision shows great sequence specificity for Cr(III)-modified DNA and Cr(III)-His-modified DNA and that the sequence specificities for both types of modified DNA are identical (8). Together, these results lead us to hypothesize that the NER system recognizes the guanine adduct, whether it is a Cr(III)-ligand-DNA or a Cr(III)-phosphate-DNA adduct, but not Cr (III)-phosphate complexes (8).…”

“…The number of UvrABC incision sites on a plasmid was calculated based on the Poisson distribution equation, P(0) = e −n , where n = number of UvrABC incisions. The data for Cr(III)-modified DNA were cited from our previous paper (8). Identification of Cr(III)-(gallate) 2 and Cr(III)-(EGA) 2 adduct formation sites in exon 5 (A), exon 7 (B), and exon 8 (C) in the p53 gene sequence by the UvrABC incision method.…”

“…Previously, we found that Escherichia coli 1 nucleotide excision repair (NER) enzymes, UvrA, UvrB, and UvrC, working in concert (termed UvrABC), are able to incise both DNA modified with histidine (His)-conjugated Cr(III) and free form Cr(III) with the same sequence specificity but incise DNA modified with the former much efficiently than the latter (8). On the basis of these results, we hypothesized that NER enzymes recognize ternary Cr(III)-DNA adducts such as Cr(III) ligand-guanine-DNA adducts and biadducts such as Cr(III)-guanine-phosphate adducts but not Cr(III)-phosphate complexes (8).…”

“…On the basis of these results, we hypothesized that NER enzymes recognize ternary Cr(III)-DNA adducts such as Cr(III) ligand-guanine-DNA adducts and biadducts such as Cr(III)-guanine-phosphate adducts but not Cr(III)-phosphate complexes (8). Consistent with our hypothesis is the recent finding from Zhitkovich's laboratory that recovery of transfected Cr(III)-cysteine-modified plasmids is much lower than that of transfected Cr(III)-modified plasmids in NER-deficient human XPA cells, a difference that is not observed in NER-proficient cells (9).…”