DNA repair and sequence context affect (1)O(2)-induced mutagenesis in bacteria.

Electronic excited molecular oxygen (singlet oxygen, (1)O(2)) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by (1)O(2) in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a (1)O(2)-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G-->T and G-->C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with (1)O(2)-induced DNA damage are linked to the context of the damaged sequence.

Mutation spectrum induced by singlet oxygen in Escherichia coli deficient in exonuclease III.

The repair of singlet oxygen (1O2)-induced DNA lesions requires several enzymes of the nucleotide and base excision repair pathways, including exonuclease III and endonuclease IV that are known apurinic/apyrimidinic-endonucleases in Escherichia coli. In order to better understand the relevance of exonuclease III on the repair of these lesions, we investigated the mutagenic events that result from the replication of a 1O2-damaged plasmid in an exonuclease-deficient host (xth). The mutation spectrum in the tRNA supF gene target indicated that the absence of exonuclease III does not change the types of mutations induced by 1O2 (mostly of G:C-->T:A and G:C-->C:G transversions). However, the spectrum shows that the mutations are scattered in the supF gene, which is significatively different from the one obtained in wild-type bacteria. Thus, exonuclease III may act on the repair of 1O2-induced lesions altering the DNA repair sequence specificity.

Inactivation of DNA proofreading obviates the need for SOS induction in frameshift mutagenesis.

Translesion synthesis at replication-blocking lesions requires the induction of proteins that are controlled by the SOS system in Escherichia coli. Of the proteins identified so far, UmuD', UmuC, and RecA* were shown to facilitate replication across UV-light-induced lesions, yielding both error-free and mutagenic translesion-synthesis products. Similar to UV lesions, N-2-acetylaminofluorene (AAF), a chemical carcinogen that forms covalent adducts at the C8 position of guanine residues, is a strong replication-blocking lesion. Frameshift mutations are induced efficiently by AAF adducts when located within short repetitive sequences in a two-step mechanism; AAF adducts incorporate a cytosine across from the lesion and then form a primer-template misaligned intermediate that, upon elongation, yields frameshift mutations. Recently, we have shown that although elongation from the nonslipped intermediate depends on functional umuDC+ gene products, elongation from the slipped intermediate is umuDC+-independent but requires another, as yet biochemically uncharacterized, SOS function. We now show that in DNA Polymerase III-proofreading mutant strains (dnaQ49 and mutD5 strains), elongation from the slipped intermediate is highly efficient in the absence of SOS induction-in contrast to elongation from the nonslipped intermediate, which still requires UmuDC functions.

New strategy for the construction of single-stranded plasmids with single mutagenic lesions.

Single-stranded DNA vectors containing single adducts offer a unique opportunity to study the biochemistry and genetics of trans lesion synthesis, a process during which a DNA polymerase synthesizes across a lesion. We describe a new and general strategy to produce high-quality single-stranded plasmids containing a single adduct within a predetermined sequence context starting with a short oligonucleotide containing the lesion of interest. These vectors are isolated from the corresponding double-stranded constructs by selective enzymatic degradation in vitro of the nonadducted uracil-containing strand. Efficient and complete removal of this strand was achieved using uracil DNA glycosilase to generate AP sites followed by the action of the AP endonuclease associated with exonuclease III and the robust 3'-->5' exonuclease activity associated with T7 DNA polymerase. We show the utility of these constructs for the study of trans lesion synthesis in vitro and in vivo in the case of the highly carcinogenic N-2-acetylaminofluorene adducts located within frameshift mutation hot spots. The possibility to construct both single-stranded and double-stranded plasmids, with the same origin of replication (i.e., ColE1), will allow a direct comparison between single-stranded and double-stranded DNA replication in site-specific mutagenesis studies.

SOS factors involved in translesion synthesis.

Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template. In Escherichia coli, the process of translesion synthesis past a lesion that hinders replication requires the induction of SOS-controlled gene products, among which are those of the umuDC operon. To study translesion synthesis in vivo, we have constructed single-stranded vectors containing single 2-acetylaminofluorene adducts located within -1 and -2 frameshift mutation hot spots formed by short repetitive sequences. These adducts strongly hinder DNA replication as only 2-5% of the molecules give rise to progeny under non-SOS-induced conditions. Induction of the SOS response lead to a 10-fold increase in survival. Adducts present within repetitive sequences trigger the formation of misaligned primer/template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis). Surprisingly we find that elongation from the nonslipped intermediate depends upon functional umuDC+ gene products, whereas elongation from the slipped intermediate is umuDC+ independent but requires another, as yet biochemically uncharacterized, SOS function. These data are discussed in terms of the different steps involved during translesion synthesis through a replication-blocking lesion.

DNA sequence determinants of carcinogen-induced frameshift mutagenesis.

In order to examine the mechanisms of induced frameshift mutagenesis, we constructed double-stranded DNA plasmids which contain single N-2-acetylaminofluorene (AAF) adducts at specified positions within a run of contiguous guanine residues. The length of the homopolymeric run and the nature of the bases flanking the contiguous sequence were systematically varied. Monomodified plasmids were introduced into SOS-induced Escherichia coli, and -1 frameshift mutations were scored by means of a phenotypic assay. A strong positional effect of the DNA adduct within the contiguous sequence was observed irregardless of the nature of the flanking bases: the AAF-induced mutation frequency was 20-200-fold higher at the 3'-end of the contiguous sequence than at the 5'-end. In addition, for a given number of guanine residues flanking the GAAF adduct on its 5'-side, differences (up to 5-fold) in the induced mutation frequency were observed as a function of the base 3' to the adduct (CGGGAAFAT approximately CGGGAAFGT > CGGGAAFCT > CGGGAAFTT). These results are discussed, within the frame of an incorporation slippage model, in terms of differences in stability and occurrence of the slipped mutagenic intermediates.

Carcinogen-induced frameshift mutagenesis in repetitive sequences.

We have constructed plasmids pS3G-1 and pSG4 that contain single acetylaminofluorene adducts within contiguous runs of three (5'-CCCG1G2G3-3') and four (5'-CG1GGG4T-3') guanine residues, respectively. In Escherichia coli, the frequency of induced -1 frameshift mutations was strongly dependent on the position of modification: pS3G-G3 was approximately 100-fold and 10-fold more mutagenic than pS3G-G1 and pS3G-G2, respectively; pSG4-G4 was approximately 600-fold more mutagenic than pSG4-G1. Mutagenesis was SOS-dependent and was markedly reduced in bacteria that were proficient in nucleotide excision repair as compared to a repair-deficient uvrA6 mutant. DNA sequencing showed that -1 frameshift events in pS3G-1 consisted of either targeted mutations (greater than 90% of induced mutations) within the guanine sequence or semitargeted mutations (greater than 10%) in the 5' flanking repetitive cytosine sequence. Semitargeted events, which were observed when acetylaminofluorene modification was at G1 and G2, show that a lesion can reduce the fidelity of replication at positions 5' to its location on the template strand. No semitargeted frameshifts were observed in plasmid pSG4, which lacks a repetitive sequence 5' to the adduct. Our results are consistent with a model for frameshift mutagenesis in which the acetylaminofluorene adduct (i) allows accurate incorporation of cytosine opposite the bulky lesion during DNA synthesis and (ii) impedes elongation of primer/template termini formed opposite the adduct or 5' to the adduct on the template strand, providing increased opportunity for the formation of slipped frameshift intermediates.