Lubomir V. Nechev

DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

ABSTRACT DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5′ to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3′-to-5′ exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.

Evaluation of the Mutagenic Potential of the Principal DNA Adduct of Acrolein

Acrolein is produced extensively in the environment by incomplete combustion of organic materials, and it arises endogenously in humans as a metabolic by-product. Acrolein reacts with DNA at guanine residues to form the exocyclic adduct, 8-hydroxypropanodeoxyguanosine (HOPdG). Acrolein is mutagenic, and a correlation exists between HOPdG levels in Salmonella typhimurium treated with acrolein and a resultant increase in mutation frequency. Site-specifically modified oligonucleotides were used to explore the mutagenic potential of HOPdG in Escherichia coli strains that were either wild-type for repair or deficient in nucleotide excision repair or base excision repair. Oligonucleotides modified with HOPdG were inserted into double-stranded bacteriophage vectors using the gapped-duplex method or into single-stranded bacteriophage vectors and transformed into SOS-induced E. coli strains. Progeny phage were analyzed by oligonucleotide hybridization to establish the mutation frequency and the spectrum of mutations produced by HOPdG. The correct base, dCMP, was incorporated opposite HOPdG in all circumstances tested. In contrast, in vitro lesion bypass studies showed that HOPdG causes misincorporation opposite the modified base and is a block to replication. The combination of these studies showed that HOPdG is not miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.

Mutagenic potential of adenine N6 adducts of monoepoxide and diolepoxide derivatives of butadiene

To determine the biological effects of specific DNA adducts resulting from the interaction of 1,3‐butadiene metabolites with DNA, deoxyoligonucleotides have been synthesized with four different adducts at the N6 position of adenine, centrally located within the human N‐ras codon 61. The adducts are those arising from adduction by either the R or S stereoisomer of the monoepoxide (BDO) or the (R,R) or (S,S) isomer of the diolepoxide (BDE). The diolepoxide can arise from partial hydrolysis of the diepoxide (BDO2) or from epoxidation of hydrolyzed monoepoxide. These adducted oligonucleotides were used in in vivo and in vitro assays designed both to determine their mutagenic potency and to examine specific interactions with Escherichia coli polymerases. Each adducted oligonucleotide was ligated into a single‐stranded vector M13mp7L2 that was subsequently used to transfect E. coli. The resulting mutagenic spectrum for these modified DNAs was stereoisomer specific. Both monoepoxide lesions were nonmutagenic, but the mutagenic spectra for the modified DNAs containing BDE adducts were stereoisomer specific. The mutations generated by adducts of the R,R enantiomer of the diolepoxide were exclusively A → G, whereas adducts of the S,S enantiomer of the diolepoxide yielded exclusively A → C mutations. None of the four modifications resulted in significant blocks to in vivo phage replication, as evidenced by no decrease in plaque‐forming ability. Consistent with these data, when each of three purified E. coli polymerases was used to replicate DNAs containing these adducted deoxyoligonucleotides, the individual polymerases appeared to be virtually unaffected, such that all lesions were readily bypassed. Whereas previous animal model studies identified the mutagenic spectrum related to butadiene exposure, these studies begin to establish the specific lesions responsible for mutagenesis. This is the first report of stereoselectivity related to butadiene‐induced mutagenesis. Environ. Mol. Mutagen. 35:48–56, 2000

Synthesis and adduction of fully deprotected oligodeoxynucleotides containing 6-chloropurine

Abstract Fully deprotected oligodeoxynucleotides containing 6-chloropurine have been synthesized and used in solution phase reactions with amine nucleophiles to prepare oligonucleotides containing substituted adenine residues. This strategy was used for the preparation of a double-stranded oligonucleotide crosslinked by a 4-carbon tether between N 6 positions of deoxyadenosines in the two strands.