Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

No abstract

DNA ligase III and the essential protein XRCC1 are present at greatly reduced levels in the xrcc1 mutant CHO cell line EM-C11. Cell-free extracts prepared from these cells were used to examine the role of the XRCC1 gene product in DNA base excision repair in vitro. EM-C11 cell extract was partially defective in ligation of base excision repair patches, in comparison to wild type CHO-9 extracts. Of the two branches of the base excision repair pathway, only the single nucleotide insertion pathway was affected; no ligation defect was observed in the proliferating cell nuclear antigen-dependent pathway. Full complementation of the ligation defect in EM-C11 extracts was achieved by addition to the repair reaction of recombinant human DNA ligase III but not by XRCC1. This is consistent with the notion that XRCC1 acts as an important stabilizing factor of DNA ligase III. These data demonstrate for the first time that xrcc1 mutant cells are partially defective in ligation of base excision repair patches and that the defect is specific to the polymerase ␤-dependent single nucleotide insertion pathway. DNA base excision repair (BER)1 counteracts the mutagenic and cytotoxic effects of various kinds of base alterations that do not significantly distort the secondary structure of the double helix. A common intermediate of this pathway is the abasic (AP) site, that arises as a consequence of removal of altered bases by DNA-N-glycosylases or as spontaneous detachment of normal bases from the deoxyribose-phosphate backbone. It has been calculated that 2000 -10000 AP sites arise each day in a mammalian cell under physiological conditions (1). Therefore, the task of BER is engaging and important, and data obtained in Escherichia coli and transgenic mice show that this process is essential for survival (2-4). We have recently shown that, in addition to the polymerase ␤-dependent single nucleotide insertion pathway previously investigated in mammalian cells (5), a distinct proliferating cell nuclear antigen (PCNA)-dependent pathway is also present that incorporates a repair patch size of 7-14 nucleotides extending 3Ј to the site of the lesion (6). Our knowledge of the enzymology of the two pathways has several gaps. In particular, the enzymology of the ligation step is poorly defined. A role for the XRCC1 protein has been suggested on the basis of the sensitivity of xrcc1 mutant cell lines (the CHO derivatives EM9 and EM-C11) to agents that introduce DNA base damage (7, 8) and because of their reduced rate of single-strand break rejoining following exposure to ionizing radiation or alkylating agents (9, 10). Consistent with a role for XRCC1 in DNA ligation and BER is its observed interaction with DNA ligase III and DNA polymerase ␤ (7, 11, 12). Here, we have examined directly the role of XRCC1 and DNA ligase III in mammalian BER using a cellfree system. We report for the first time that (i) xrcc1 mutant cells are partially defective in ligation of BER patches and (ii) the defect involves only the polymerase ␤-dependent single nucle...

No abstract

No abstract

Glyphosate (N-phosphonomethylglycine) is an effective herbicide acting on the synthesis of aromatic amino acids in plants. The genotoxic potential of this herbicide has been studied: the results available in the open literature reveal a weak activity of the technical formulation. In this study, the formulated commercial product, Roundup, and its active agent, glyphosate, were tested in the same battery of assays for the induction of DNA damage and chromosomal effects in vivo and in vitro. Swiss CD1 mice were treated intraperitoneally with test substances, and the DNA damage was evaluated by alkaline elution technique and 8-hydroxydeoxyguanosine (8-OHdG) quantification in liver and kidney. The chromosomal damage of the two pesticide preparations was also evaluated in vivo in bone marrow of mice as micronuclei frequency and in vitro in human lymphocyte culture as SCE frequency. A DNA-damaging activity as DNA single-strand breaks and 8-OHdG and a significant increase in chromosomal alterations were observed with both substances in vivo and in vitro. A weak increment of the genotoxic activity was evident using the technical formulation.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N 3 -methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT 3 TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A 600 -602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n ‫؍‬ 9, p < 10 ؊6 , Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: ⌬mag1, ⌬apn1apn2, and ⌬apn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in ⌬mag1, ⌬apn1apn2, and ⌬apn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < ⌬mag1< ⌬apn1apn2 ‫؍‬ ⌬apn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in ⌬mag1, and 23 in ⌬apn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in ⌬mag1, and 23 in ⌬apn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in ⌬mag1; and 14/23, 61%, in ⌬apn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.Most alkylating agents react with nucleophilic sites on DNA, yielding a complex mixture of DNA lesions (1). Thus, quantitative and qualitative analyses of the biological role(s) of individual DNA lesions and their relative contribution to the mutagenicity and/or toxicity are difficult tasks. Many clinically used anticancer drugs are DNA alkylating agents. Hence, an understanding of which biological effect is caused by a specific DNA lesion may lead to a more rational design of antineoplastic agents.To exercise a significant regulation over the alkylation pattern on DNA, several alkylating agents were recently synthesized, including Me-lex, 1 a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide (lex)